Synergistic combinations of ox40l antibodies for the treatment of gvhd

ABSTRACT

The present invention relates to anti-human OX40L antibodies, new medical uses and methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. §120 ofco-pending U.S. application Ser. No. 15/142,538 filed on Apr. 29, 2016,the contents of which are in incorporated herein by reference in theirentireties.

U.S. application Ser. No. 15/142,538 filed on Apr. 29, 2016, is acontinuation application under 35 U.S.C. §120 of co-pendingInternational Application No. PCT/GB2016/050565 filed Mar. 3, 2016,which designated the U.S., and which claims priority ofPCT/GB2015/050614 filed on Mar. 3, 2015, which claims priority to GBPatent Application No. GB1516008.8 filed on Sep. 9, 2015 the contents ofeach of which are incorporated herein by reference in their entireties.

U.S. application Ser. No. 15/142,538 filed on Apr. 29, 2016, is aContinuation-in-Part of co-pending application under 35 U.S.C. §120 ofco-pending International Application No. PCT/GB2015/050614 filed on Mar.3, 2015, which claims priority to GB Patent Application No. GB1516008.8filed on Sep. 9, 2015 the contents of each of which are incorporatedherein by reference in their entireties.

U.S. application Ser. No. 15/142,538 filed on Apr. 29, 2016, is aContinuation-in-Part application of U.S. application Ser. No. 14/935,937filed on Nov. 9, 2015, now U.S. Pat. No. 9,434,785 issued Sep. 6, 2016,which is a Continuation-in-Part application of U.S. application Ser. No.14/811,163 filed on Jul. 28, 2015, now U.S. Pat. No. 9,234,043 issuedJan. 12, 2016, which is a continuation of U.S. application Ser. No.14/700,896, filed on Apr. 30, 2015, now U.S. Pat. No. 9,139,653, issuedon Sep. 22, 2015. This application also claims priority to GB PatentApplication No. GB1516008.8 filed on Sep. 9, 2015 the contents of eachof which are incorporated herein by reference in their entireties.

U.S. application Ser. No. 15/142,538 filed on Apr. 29, 2016, is aContinuation-in-Part application under 35 U.S.C. §120 of U.S.application Ser. No. 14/955,843 filed on Dec. 1, 2015, now abandoned,which is a Continuation application of U.S. application Ser. No.14/811,163 filed on Jul. 28, 2015, now U.S. Pat. No. 9,234,043 issuedJan. 12, 2016, which is a continuation of U.S. application Ser. No.14/700,896, filed on Apr. 30, 2015, now U.S. Pat. No. 9,139,653, issuedon Sep. 22, 2015. This application also claims priority to GB PatentApplication No. GB1516008.8 filed on Sep. 9, 2015 the contents of eachof which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 25, 2016, isnamed Sequence_listing-069496-087031.txt and is 184,690 bytes in size.

TECHNICAL FIELD

The present invention relates to anti-human OX40L antibodies, newmedical uses and methods.

BACKGROUND

OX40 ligand (OX40L) is a TNF family member; a 34 kDa type IItransmembrane protein. The crystallized complex of human OX40 and OX40Lis a trimeric configuration of one OX40L (trimer) and three OX40monomers. The human extracellular domain is 42% homologous to mouseOX40L.

OX40L is not constitutively expressed but can be induced on professionalAPCs such as B-cells, dendritic cells (DCs) and macrophages. Other celltypes such as Langerhans cells, endothelial cells, smooth muscle cells,mast cells and natural killer (NK) cells can be induced to expressOX40L. T-cells can also express OX40L. The OX40L receptor, OX40, isexpressed on activated T-cells (CD4 and CD8 T-cells, Th2, Th1 and Th17cells) and CD4⁺Foxp3⁺ cells, even in the absence of activation.

The interaction between OX40 and OX40L occurs during the T-cell-DCinteraction 2 or 3 days after antigen recognition. After leaving DCs,the OX40-expressing T-cell may interact with an OX40L-expressing cellother than a DC and receive an OX40 signal from this cell, which mayprovide essential signals for the generation of memory T-cells, theenhancement of Th2 response and the prolongation of the inflammatoryresponses. OX40 signals into responder T-cells render them resistant toTreg mediated suppression.

Graft versus host disease is a major cause of mortality followingallogeneic bone marrow treatment. In the acute version of the disease,mature T-cells present in the bone marrow graft recognise the donortissue as foreign in an environment of damaged tissue, which, via hostAPC's cause the activation and proliferation of the donor T-cells, withsubsequent T-cell migration into the liver, spleen, gut, skin and lungs,causing tissue damage by the CTL effector response and inflammatorycytokine/chemokine release. Onset for acute disease is usually withinthe first 100 days post transplantation (Hill-Ferrara, Blood May 1, 2000vol. 95 no. 9 2754-275, Reddy-Ferrara Blood, Volume 17, Issue 4,December 2003).

Chronic GvHD usually appears 100 days post transplantation and severalfactors are thought to be involved, including thymic damage caused byprior acute GvHD which results in a reduced clearance of pathogenicT-cells (Zhang et al, Sep. 1, 2007 vol. 179 no. 5 3305-3314),up-regulation of TGF-β, which causes fibrosis (McCormick et al J Immuno,Nov. 15, 1999 vol. 163 no. 10 5693-5699), and a B-cell component drivenby elevated B-Cell activating factor (BAFF) (Sarantopoulos et al, ClinCancer Res Oct. 15, 2007 13; 6107) as well as auto-antibodies againstplatelet derived growth factor receptor (Svegliati et al, Blood Jul. 1,2007 vol. 110 no. 1 237-241).

Clinical studies have shown that OX40 is up-regulated in both acute(Morante et al, Clinical and Experimental Immunology, 145:36-43) andchronic (Kotani et al, Blood Nov. 15, 2001 vol. 98 no. 10 3162-3164)GvHD. Administration of an antagonistic anti-OX40L enhanced survival ina lethal acute mouse model of GvHD, with a 70% survival in the treatedgroup compared to the untreated who all died by day 43 (Tsukada et al,Blood, 1 Apr. 2000, Volume 95, Number 7) whereas treatment with anagonistic anti-OX40 Ab accelerated the disease and mortality (Blazar etal Blood May 1, 2003 vol. 101 no. 9 3741-3748). Blockade of theOX40-OX40L interaction has been shown to be efficacious in several otherinflammatory disease, with anti-OX40L Ab being used to treat a mousemodel of colitis (Totsuka et AJP-GI Apr. 1, 2003 vol. 284 no. 4G595-G603), and that an anti-OX40L Ab could block the development ofdiabetes in NOD mice (Pakala et al European Journal of Immunology Volume34, Issue 11, pages 3039-3046, November 2004).

REFERENCES

-   Lamb, L. S., Abhyankar, S. A., Hazlett, L., O'Neal, W., Folk, R. S.,    Vogt, S., Parrish, R. S., Bridges, K., Henslee-Downey, P. J. and    Gee, A. P. (1999), Expression of CD134 (OX-40) on T-cells during the    first 100 days following allogeneic bone marrow transplantation as a    marker for lymphocyte activation and therapy-resistant    graft-versus-host disease. Cytometry, 38: 238-243.-   Xupeng Ge, Julia Brown, Megan Sykes, Vassiliki A. Boussiotis,    CD134-Allodepletion Allows Selective Elimination of Alloreactive    Human T-cells without Loss of Virus-Specific and Leukemia-Specific    Effectors, Biology of Blood and Marrow Transplantation, Volume 14,    Issue 5, May 2008, Pages 518-530.-   Naoto Ishii, Takeshi Takahashi, Pejman Soroosh, Kazuo Sugamura,    Chapter 3—OX40-OX40 Ligand Interaction in T-Cell-Mediated Immunity    and Immunopathology, In: Frederick W. Alt, Editor(s), Advances in    Immunology, Academic Press, 2010, Volume 105, Pages 63-98.-   Croft, M., So, T., Duan, W. and Soroosh, P. (2009), The significance    of OX40 and OX40L to T-cell biology and immune disease.    Immunological Reviews, 229: 173-191.

SUMMARY OF THE INVENTION

The invention provides anti-human OX40L (hOX40L) antibodies andfragments and novel medical applications for treating or preventinghOX40L-mediated diseases or conditions in humans. To this end, theinvention provides:—

In a First Configuration

An antibody or a fragment thereof that specifically binds to hOX40L fortreating or preventing a hOX40L-mediated disease or condition in a humanin a method wherein the antibody or fragment is administered to saidhuman, wherein the antibody or fragment is for treating or preventingsaid hOX40L-mediated disease or condition by decreasing one, more or allof

-   -   a. secretion of a cytokine selected from TNF alpha, IL-2, IL-3,        IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES and        interferon gamma in the human;    -   b. the proliferation of leukocytes of the human; and    -   c. binding of hOX40 receptor expressed by human T-cells with        endothelial cell expressed hOX40L.

In a Second Configuration

An antibody or a fragment thereof, that specifically binds to hOX40L andcompetes for binding to said hOX40L with an antibody selected from thegroup consisting of 02D10, 10A07, 09H04 and 19H01.

In a Third Configuration

Use of an antibody or a fragment thereof, that specifically binds tohOX40L in the manufacture of a medicament for administration to a human,for treating or preventing a hOX40L-mediated disease or condition in thehuman by decreasing one, more or all of

-   -   a. secretion of a cytokine selected from TNF alpha, IL-2, IL-3,        IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES and        interferon gamma in the human;    -   b. the proliferation of leukocytes of the human; and    -   c. binding of hOX40 receptor expressed by human T-cells with        endothelial cell expressed hOX40L.

In a Fourth Configuration

A method of treating or preventing a hOX40L-mediated disease orcondition in a human by decreasing one, more or all of

-   -   a. secretion of a cytokine selected from TNF alpha, IL-2, IL-3,        IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES and        interferon gamma in the human;    -   b. the proliferation of leukocytes of the human; and    -   c. binding of hOX40 receptor expressed by human T-cells with        endothelial cell expressed hOX40L;    -   wherein the method comprises administering to said human a        therapeutically effective amount of an antibody or fragment that        specifically binds to hOX40L.

In a Fifth Configuration

An antibody or a fragment thereof, that specifically binds to hOX40L andcompetes for binding to said hOX40L with the antibody 02D10, wherein theantibody or fragment comprises a VH domain which comprises a HCDR3comprising the motif VRGXYYY, wherein X is any amino acid.

In a Sixth Configuration

An antibody or a fragment thereof, that specifically binds to hOX40L andcompetes for binding to said hOX40L with the antibody 02D10, wherein theantibody or fragment comprises a VH domain which comprises the HCDR3sequence of SEQ ID NO:40 or 46 or the HCDR3 sequence of SEQ ID NO:40 or46 comprising less than 5 amino acid substitutions.

In a Seventh Configuration

A human antibody or fragment thereof comprising a HCDR3 of from 16 to 27amino acids and derived from the recombination of a human VH genesegment, a human D gene segment and a human JH gene segment, wherein thehuman JH gene segment is IGHJ6, which specifically binds to hOX40L fortreating or preventing an autoimmune disease selected from an autoimmunedisease or condition, a systemic inflammatory disease or condition, ortransplant rejection.

In an Eighth Configuration

Use of a human antibody or fragment thereof comprising a HCDR3 of from16 to 27 amino acids and derived from the recombination of a human VHgene segment, a human D gene segment and a human JH gene segment,wherein the human JH gene segment is IGHJ6, which specifically binds tohOX40L in the manufacture of a medicament for administration to a humanfor treating or preventing a hOX40L mediated disease or condition in thehuman selected from an autoimmune disease or condition, a systemicinflammatory disease or condition, or transplant rejection.

In a Ninth Configuration

A method of treating or preventing a hOX40L mediated disease orcondition selected from an autoimmune disease or condition, a systemicinflammatory disease or condition, or transplant rejection, comprisingadministering to said human a therapeutically effective amount of ahuman antibody or fragment thereof comprising a HCDR3 of from 16 to 27amino acids and derived from the recombination of a human VH genesegment, a human D gene segment and a human JH gene segment, wherein thehuman JH gene segment is IGHJ6, which specifically binds to hOX40L,wherein the hOX40L mediated disease or condition is thereby treated orprevented.

The invention also provides pharmaceutical compositions, kits, nucleicacids, vectors and hosts.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows profiling of fully human recombinant anti-OX40L antibodiesin HTRF Ligand/Receptor Neutralisation assay. Data shown isrepresentative of three repeat experiments.

FIGS. 2A-2F show determining effect of anti-OX40L antibodies inallogeneic PBMC/T Mixed Lymphocyte Reaction. Data shown is from threeindependent donor pairings where it is assumed each donor is a differentindividual. FIG. 2A shows the effect of anti-OX40L antibodies in PBMC/TMLR percentage inhibition relative to no IgG wells for donor pairing 1;FIG. 2B shows the same for donor pairing 2; and FIG. 2C for donorpairing 3. FIG. 2D shows the effect of anti-OX40L antibodies in PBMC/TMLR IFN gamma relative to no IgG wells for donor pairing 1; FIG. 2Eshows the same for donor pairing 2 and FIG. 2F shows the same for donorpairing 3.

FIGS. 3A-3B show expansion of Tscm cells following allogeneic HCT. Plotsare gated on CD4+CD45RA+CCR7+ T-cells. FIG. 3A shows pre-HCT and FIG. 3Bshows post-HCT cell distribution.

FIGS. 4A-4B show OX40L blockade controls expansion of CD4+ Tscm cellswhile preserving CD4+ T naïve cells following allogeneic HCT. Absolutenumbers of peripheral blood CD4+ Tscm (FIG. 4A) and Tnaive (FIG. 4B)following allogeneic HCT in control animal and 2D10 IgG4PE treatedanimals.

FIGS. 5A-5C show OX40 expression on naïve CD4+ T and memory stem T-cellsin a representative animal following allogeneic HCT. FIGS. 5A and 5Bshow FACS plots were gated on CD3+CD4+CD45RA+CCR7+. The histogram ofFIG. 5C shows OX40 expression in different T-cell subsets of CD4+T-cells: Naïve (CD45RA+CCR7+CD95−), memory stem (SCM:CD45RA+CCR7+CD95+), central memory (CM: CD45RA-CCR7+), effector memory(EM: CD45RA-CCR7−) and terminally differentiated effector-memory cellsre-expressing CD45RA (TEMRA: CD45RA+CCR7−).

FIG. 6A shows effects of OX40L blockade on SCM CD4+ T-cells. Datapointsfor 02D10 Ig4PE are shown by circles, rapamycin by filled squares andtacrolimus plus methotrexate (Tac/MTX) by open squares.

FIG. 6B shows effects of OX40L blockade on SCM CD8+ T-cells. Datapointsfor 02D10 Ig4PE are shown by circles, rapamycin by filled squares andTac/MTX by unfilled squares.

FIG. 7 shows Kaplan-Meier survival curve for rhesus monkey recipients ofhematopoietic stem cell transplants derived from the peripheral blood ofhaploidentical half-sibling donors. Results are shown for animals thatdid not receive post-transplant prophylactic therapy (No-treatmentcontrol; median survival time, MST=8 days; n=4), and those receivingrapamycin monotherapy (MST=17 days; n=4), 2D10 monotherapy (MST=19 days;n=4), or rapamycin plus 2D10 (MST>82 days; n=3). Note that animals 2 and3 indicated on the figure were on-study at the time of drafting; neithershowed signs of GVHD at Day 82 or Day 41 post-transplant, respectively.Asterix * for the no treatment control and rapamycin monotherapy groupsis data taken from Furlan et al., 2015, Science Translational Medicine,vol 7 (315), 315ra191.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides the following aspects 1 to 113.

The invention is useful, for example, for treating or preventingtransplant rejection, e.g., graft versus host disease (GvHD) orallogeneic transplant rejection. The invention is also useful, forexample, for treating or preventing an inflammatory bowel disease, e.g.,UC or CD, or for treating or preventing an airway inflammatory diseaseor condition. In an example this aspect is useful for treating orpreventing asthma. The invention is also useful, for example, fortreating or preventing fibrosis. The invention is also useful, forexample, for treating or preventing diabetes. The invention is alsouseful, for example, for treating or preventing uveitis. The inventionis also useful, for example, for treating or preventing pyodermagangrenosum. The invention is also useful, for example, for treating orpreventing giant cell arteritis. The invention is also useful, forexample, for treating or preventing Schnitzler syndrome. The inventionis also useful, for example, for treating or preventing non-infectiousscleritis.

-   1. An antibody or a fragment thereof that specifically binds to    hOX40L for treating or preventing a hOX40L-mediated disease or    condition in a human in a method wherein the antibody or fragment is    administered to said human, wherein the antibody or fragment is for    treating or preventing said hOX40L-mediated disease or condition by    decreasing one, more or all of    -   a. secretion of a cytokine selected from TNF alpha, IL-2, IL-3,        IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES and        interferon gamma in the human;    -   b. the proliferation of leukocytes of the human; and    -   c. binding of hOX40 receptor expressed by human T-cells with        endothelial cell expressed hOX40L.

The inventors, thus identified for the first time decreases of (a), (b)and (c) as ways of treating and/or preventing OX40L-mediated disease andconditions in humans and they provide antibodies and antibody fragmentsfor this purpose.

In an example, the secretion is leukocyte secretion. In an example, (a)is indicated by a significantly elevated level of the cytokine(s) inhuman blood, plasma or serum.

In an example, the cytokine is selected from (i) TNF alpha, (ii) IL-2and (iii) interferon gamma. In example, the cytokine TNF alpha. Inexample, the cytokine is IL-2. In an example, the cytokine is interferongamma. In an example, the cytokines are (i) and (ii); or (i) and (iii);or (ii) and (iii); or (i)-(iii).

In an example, the decrease of (a), (b) or (c) or any other decreasedisclosed herein is a decrease of at least 10 or 20% compared to thelevel in a human at risk of or suffering from the hOX40L-mediateddisease or condition. In an example, the latter is the human recited inaspect 1 prior to administration of the antibody or fragment; in anotherexample the latter human is a different human. In an example, saiddecrease is at least 10, 20, 30, 40, 50 or 60%.

-   -   (i) In an example, the antibody or fragment is capable of        effecting a decrease of secretion of the relevant cytokine from        leukocytes (eg, human T-cells) in an in vitro assay (as        explained further below), and thus administration of such        antibody or fragment to the human leads to decrease of (a).    -   (ii) In an example, the antibody or fragment is capable of        effecting a decrease of the proliferation of leukocytes (eg,        human PBMCs and/or human T-cells) in an in vitro assay (as        explained further below), and thus administration of such        antibody or fragment to the human leads to decrease of (b).    -   (iii) In an example, the antibody or fragment is capable of        effecting a decrease of the binding of hOX40 receptor expressed        by human T-cells with endothelial cell expressed hOX40L in an in        vitro assay (as explained further below), and thus        administration of such antibody or fragment to the human leads        to decrease of (c).

In an example, (i) and (ii); or (i) and (iii); or (ii) and (iii); or(i)-(iii) apply.

Additionally or alternatively, assessment of said decreases can beperformed using samples from the treated human. For example, referenceis made to J. Clin. Immunol., 2004 January, 24(1):74-85; “Increasedexpression of CCL20 in human inflammatory bowel disease”; Kaser A et al.This publication provides an example of a generally-applicable techniqueof using tissue biopsies and reading out decreased cytokine levelsindicative of decreased cytokine secretion after treatment with anantibody in vivo. Similar methods can be used to determine decrease ofthe secretion of one or more cytokines in a human having received anantibody of the invention. The skilled person will be familiar withtechniques for assessing cytokine levels in patients and patientsamples, for example, by use of one or more of tissue biopsy,immunohistochemistry, immunofluorescence, tissue staining, cytokine mRNAquantification (e.g., using PCR, such as Taqman™ PCR), cytokine proteindetection and quantification (e.g., using cytokine-specific toolantibody and quantification, such as by ELISA or another standardprotein quantification technique). For example, where the disease orcondition is one of the GI tract (e.g., IBD), one can perform biopsy ofrelevant gut tissue from a patient that has received an antibody of theinvention, followed by quantification of cytokine mRNA and/or cytokineprotein (e.g., using quantitative PCR). The result can be compared witha cytokine quantification in biopsied relevant tissue from the samepatient prior to antibody administration or compared to another humanpatient suffering from the same disease or condition but receiving noanti-OX40L treatment or no treatment for the disease or condition. Inthis way, the skilled person can determine that the antibody of theinvention decreases secretion of the cytokine in the human recipient.Instead of assessing gut tissue levels, one can instead use a differenttissue or sample from the human patient dependent upon the nature andlocation of the disease or condition. For example, where the disease orcondition is one of the airways (e.g., lung), it is possible to take alung or other airway tissue sample for cytokine assessment.Alternatively, one can use a Bronchoalveolar lavage (BAL) sample, aswill be apparent to the skilled person. In another example, for somedisease or conditions one can assess the decrease in cytokine in ablood, serum or plasma sample taken from a human that has received anantibody of the invention, and then comparing to the level beforereceiving the antibody or comparing to the level in an untreated human,as discussed above.

As is known in the art, the term “leukocytes” includes, for example, oneor more of lymphocytes, polymorphonuclear leukocyte and monocytes. As isalso readily apparent to the skilled person the term “monocytes”includes, for example, peripheral blood mononuclear cells (PBMCs) ormonocyte derived cells, e.g., dendritic cells (DCs). See, for example,Immunobiology, 2013 November, 218(11):1392-401. doi:10.1016/j.imbio.2013.07.005. Epub 2013 Jul. 25; “Leukoreduction systemchambers are an efficient, valid, and economic source of functionalmonocyte-derived dendritic cells and lymphocytes”, Pfeiffer I A et al.

The proliferation of leukocytes, e.g., lamina propria lymphocytes(LPLs), can be assessed using tissue biopsy, staining and histology, aswill be apparent to the skilled person. Hematoxylin and eosin stain (H&Estain or HE stain) is, for example, commonly used in histology to lookfor infiltrating lymphocytes a whole range of human tissue and is one ofthe principal stains in histology. It is the most widely used stain inmedical diagnosis and is often the gold standard, and as such can beused to assess proliferation of leukocytes as per the invention. Forexample, GI tract tissue (e.g., gut tissue) from a human that issuffering from or at risk of a hOX40L-mediated disease or condition canbe obtained, stained and assessed for the extent of infiltration ofLPLs. Comparison can be made between such tissue from a human that hasreceived an antibody of the invention compared to the extent ofinfiltration in tissue obtained from the same human prior toadministration of antibody or from another human that has not receivedtreatment and is at risk of or suffering from the disease or condition.For example, the comparison is between human gut tissues taken from thesame (or different) humans suffering from IBD.

One can, for example, determine if the antibody or fragment is capableof decreasing binding of hOX40 receptor expressed by human T-cells withendothelial cell expressed hOX40L using standard binding assays arefamiliar to the skilled person, e.g., using ELISA or SPR.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorderaffecting the gastrointestinal tract with an apparently ever-increasingincidence and tendency to more severe clinical phenotypes. The diseaseis characterised by an exaggerated immune response to the luminal flora,suggesting that deficiencies in barrier function of intestinal flora maybe involved, and studies support this notion (Cucchiara et al., 2012;Jostins et al., 2012; Manichanh et al., 2012; Salzman et al., 2007, allcited in Deuring et al., “The cell biology of the intestinal epitheliumand its relation to inflammatory bowel disease”, The InternationalJournal of Biochemistry & Cell Biology 45 (2013) 798-806). IBD includestwo main groups: Crohn's disease (CD) and ulcerative colitis (UC). CDpatients can have inflammatory lesions in their entire gastrointestinaltract, whereas the inflammation in UC patients is restricted to thecolon. Reference is also made to Hisamatsu et al. (“Immune aspects ofthe pathogenesis of inflammatory bowel disease”, Pharmacology &Therapeutics 137 (2013) 283-297) and the documents cited therein.

Granuloma formation is the one of the most important pathologicalcharacteristics of human Crohn's disease. Mizoguchi et al demonstratedthat F4/80-positive immature CD11c⁺ dendritic cells (DCs) produce IL-23and contribute to granuloma formation in a murine colitis model(Mizoguchi et al., 2007). A Th1 immune response is predominant inCrohn's disease. Indeed, CD4⁺ T-cells in the LP of Crohn's diseaseexpressed T-bet and produced large amounts of interferon (IFN)-γ(Matsuoka et al., 2004). Sakuraba et al demonstrated that DCs in themesentric lymph nodes of patients with Crohn's disease strongly promoteda Th1 and Th17 immune response (Sakuraba et al., 2009). Mesentric lymphnode DCs contribute to IBD pathogenesis, particularly that of Crohn'sdisease.

Role of Cytokines in Disease and Conditions

Reference is made to Muzes et al, World J Gastroenterol 2012 Nov. 7;18(41): 5848-5861 ISSN 1007-9327 (print) ISSN 2219-2840 (online),“Changes of the cytokine profile in inflammatory bowel Diseases”.

Cytokines are indispensable signals of the mucosa-associated immunesystem for maintaining normal gut homeostasis. An imbalance of theirprofile in favour of inflammation initiation may lead to disease states,such as that is observed in inflammatory bowel diseases (IBD), e.g.,Crohn's disease (CD) and ulcerative colitis (UC). The role ofpro-inflammatory cytokines such as IL-1α, IL-1β, IL-2, -6, -8, -12, -17,-23, IFN-gamma, or TNF alpha in TBD is associated with the initiationand progression of UC and CD. CD is often described as a prototype ofT-helper (Th) 1-mediated diseases because the primary inflammatorymediators are the Th1 cytokines such as interleukin (IL)-12, interferon(IFN)-γ, and tumour necrosis factor (TNF)-α.

Binding of TNF-like ligands to their receptors triggers intracellularpathways that are directly involved in cell proliferation,differentiation, and survival. Most members of the TNF/TNF-receptorprotein superfamilies are expressed on immune cells and play a criticalrole in multiple components of the immune response. TNF-α is a mastercytokine in the pathogenesis of IBD. It exerts its pleiotropic effectsthrough the expression of adhesion molecules, fibroblast proliferation,procoagulant factors, as well as the initiation of cytotoxic, apoptoticand acute-phase responses. The source of TNF-α in IBD is partly theinnate immune cells, such as macrophages or monocytes, and alsodifferentiated Th1 cells. The serum levels of TNF-α correlate with theclinical activity of UC and CD[31]. It plays an orchestrating role incolonic inflammation in IBD. The role of TNF-α in CD has been widelyinvestigated. Binding TNF-α to serum soluble TNF receptor 1 and 2(sTNFR1 and 2) initiates pro-inflammatory signalling. The levels ofsTNFR1 and 2 are elevated in CD.

Tumour necrosis factor-like factor (TL1A), another member of the TNFfamily, stimulates TFN-γ secretion by binding to death receptor 3 (DR3).DR3 is expressed by a high percentage of cells from mucosal biopsies ofUC and CD, and an increase of IFN-γ level has been observed with diseaseactivity in IBD patients. The TL1A/DR3 system is involved in thepathogenesis of CD. The macrophages of the lamina propria are a majorproducer of TL1A, which expression is markedly enhanced in CD. It hasbeen found that TL1A and IL-23 synergistically promotes the productionof IFN-γ by mucosal T-cells. FN-Y: is produced by TH1 T-cells. Onceinflammation is initiated, IFN-γ is produced and subsequently actsthrough various molecules and pathways of the immune system to intensifythe inflammatory process. There is an overwhelming body of literatureextensively documenting the proinflammatory nature of IFN-γ which hasled to the mainstream opinion that IFN-γ is a prime proinflammatorycytokine in inflammation and autoimmune disease. Interferon-gamma iscausatively involved in experimental inflammatory bowel disease in mice(Ito et al, Clinical and Experimental Immunology (2006), 146:330-338).The study clearly demonstrated that IFN-γ^(−/−) mice manifestedattenuated colitis after stimulation with DSS, in terms of the degree ofbody weight loss, DAI, histological score and MPO activity. IFN-γ wasincreasingly produced in the colon of DSS-treated WT mice that showedsevere IBD-like symptoms.

Interleukin-2 (IL-2) is produced by T-cells and is mostly important forT-cells to differentiate into effector T-cells. IL-2 is also importantfor T-cell proliferation. This is important for IBD because effectorT-cells are thought to be a major cell type to cause damage in IBD.

IL-8 (interleukin-8; aka CXCL8) primarily mediates the activation andmigration of neutrophils into tissue from peripheral blood and to sitesof inflammation. The tissue level of IL-8 has been found to be higher inactive UC compared to normal colonic tissue, and its serum concentrationhas been related to endoscopic and histological severity of UC. IL-8 isimportant for inflammatory settings and cancer (sec, e.g., “TheChemokine CXCL8 in Carcinogenesis and Drug Response”, ISRN Oncol. 2013Oct. 9; 2013:859154; Gales D et al., and Future Oncol., 2010 January;6(1):111-6. doi: 10.2217/fon.09.128; “CXCL8 and its cognate receptors inmelanoma progression and metastasis”, Singh S et al.). In cancerparticularly, IL-8 is thought to contribute also by supportingangiogenesis.

In any configuration, aspect, concept or example herein the antibody orfragment antagonises the binding of hOX40L to an OX40 receptor.

In any configuration, aspect, concept or example herein, the antibody orfragment antagonises the binding of hOX40L to OX40.

In any configuration, aspect, concept or example herein, the OX40Lreceptor can be human OX40.

In any configuration, aspect, concept or example herein the human issuffering from or at risk of asthma and the antibody or fragmentdecreases IgE in a human.

In any configuration, aspect, concept or example herein the human issuffering from or at risk of asthma and the antibody or fragment is fordecreasing IgE in a human.

-   2. The antibody or fragment of aspect 1, wherein the antibody or    fragment decreases the binding of hOX40 receptor expressed by human    T-cells with endothelial cell expressed hOX40L and decreases the    proliferation of human T-cells; wherein the antibody or fragment is    for treating or preventing said hOX40L-mediated disease or condition    by decreasing the secretion of a cytokine selected from TNF alpha,    IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17,    RANTES and interferon gamma.

In an example, the cytokine is selected from (i) TNF alpha, (ii) IL-2and (iii) interferon gamma. In an example, the cytokine is TNF alpha. Inan example, the cytokine is IL-2. In an example, the cytokine isinterferon gamma. In an example, the cytokines are (i) and (ii); or (i)and (iii); or (ii) and (iii); or (i)-(iii).

-   3. The antibody or fragment of aspect 1, wherein the leukocytes are    selected from the group consisting of polymorphonuclear leukocytes,    monocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes,    T-cells, antigen presenting cells (APCs), dendritic cells (DC cells)    and natural killer cells (NK cells).

In one embodiment, the leukocytes are peripheral blood mononuclear cells(PBMCs) and T-cells (e.g. PBMCs).

-   4. The antibody or fragment of aspect 3, wherein the leukocytes    comprise lamina propria lymphocytes (LPLs) and the disease or    condition is a disease or condition of the gastrointestinal tract    (GI tract).-   5. The antibody or fragment of any preceding aspect, wherein the    epithelial cells comprise cells selected from the group consisting    of gastrointestinal cells, colon cells, intestinal cells and airway    (e.g., lung) epithelial cells.

In another embodiment, the epithelial cells comprise cells selected fromthe group consisting of gastrointestinal cells, colon cells, intestinalcells, ocular cells and airway (e.g., lung) epithelial cells. In anotherembodiment, the epithelial cells comprise cells selected from the groupconsisting of gastrointestinal cells, colon cells, intestinal cells andocular cells. In a further embodiment, the epithelial cells compriseocular cells.

-   6. The antibody or fragment of any preceding aspect, for treating or    preventing said hOX40L-mediated disease or condition in said human    by decreasing the proliferation of T-cells in said human.

In an example, the antibody or fragment is capable of effecting adecrease of the proliferation of T-cells in an in vitro assay (e.g., ina human DC cell/T-cell in vitro assay, for example as explained furtherbelow), and thus administration of such antibody or fragment to thehuman leads to decrease of the proliferation of T-cells in said human.

-   7. The antibody or fragment of any preceding aspect, for treating or    preventing said hOX40L-mediated disease or condition in said human    by antagonising the interaction between hOX40L and leukocytes of the    human, wherein the proliferation of leukocytes is decreased.

In an example, the antibody or fragment is capable of effecting adecrease of the proliferation of leukocytes (e.g., mononuclear cells) inan in vitro assay (e.g., in a MLR in vitro assay, for example asexplained further below), and thus administration of such antibody orfragment to the human leads to decrease of the proliferation ofleukocytes in said human.

-   8. The antibody or fragment of any preceding aspect, for treating or    preventing said hOX40L-mediated disease or condition in said human    by decreasing the proliferation of leukocytes of the human by    antagonising the OX40L/OX40L receptor interaction mediated by    T-cells in said human.

In an example, the antibody or fragment is capable of effecting adecrease of the proliferation of leukocytes (e.g., mononuclear cells) inan in vitro assay wherein the antibody or fragment antagonisesOX40L/OX40L receptor interaction mediated by T-cells in said assay, andthus administration of such antibody or fragment to the human leads todecrease of the proliferation of leukocytes in said human.

-   9. The antibody or fragment of any preceding aspect, for treating or    preventing said hOX40L-mediated disease or condition in said human    by decreasing the secretion of a cytokine selected from TNF alpha,    IL-2 and interferon gamma in the human.

In an example, the antibody or fragment is for treating or preventingsaid hOX40L-mediated disease, condition or epithelial cell damage insaid human by decreasing the secretion of (i) IL-2 and interferon gamma,(ii) IL-2 and TNF alpha or (iii) interferon gamma and TNF alpha in thehuman.

In an example, the antibody or fragment is capable of effecting adecrease of the secretion of a cytokine selected from IL-2, TNF alphaand interferon gamma in an in vitro assay (e.g., in a MLR in vitroassay, for example as explained further below), and thus administrationof such antibody or fragment to the human leads to decrease of thesecretion of said selected cytokine(s) in said human.

In an example, the antibody or fragment is capable of effecting adecrease of the secretion of IL-8 in an in vitro assay (e.g., in a MLRin vitro assay, for example as explained further below), and thusadministration of such antibody or fragment to the human leads todecrease of the secretion of IL-8 in said human.

-   10. The antibody or fragment of aspect 9, for treating or preventing    said disease or condition by decreasing the secretion of said    cytokine mediated by the interaction of dendritic cells (DC cells)    with T-cells in the human.

In an example, the antibody or fragment is capable of effecting adecrease of said cytokine(s) secretion in a DC cell/T-cell in vitroassay (for example as explained further below), and thus administrationof such antibody or fragment to the human leads to decrease of thesecretion of said cytokine(s) in said human.

-   11. The antibody or fragment of any preceding aspect, wherein    gastrointestinal cell, colon cell, intestinal cell or airway (e.g.,    lung) cell damage is a symptom or cause of said disease or condition    in humans.

In another embodiment, the epithelial cells comprise cells selected fromthe group consisting of gastrointestinal cells, colon cells, intestinalcells, ocular cells and airway (e.g., lung) epithelial cells. In anotherembodiment, the epithelial cells comprise cells selected from the groupconsisting of gastrointestinal cells, colon cells, intestinal cells andocular cells. In a further embodiment, the epithelial cells compriseocular cells.

-   12. The antibody or fragment of any preceding aspect, wherein the    human is suffering from or at risk of an inflammatory bowel disease    (IBD), allogeneic transplant rejection, graft-versus-host disease    (GvHD), diabetes or airway inflammation and said method treats or    prevents IBD, allogeneic transplant rejection, GvHD, diabetes or    airway inflammation in the human.-   12a. The antibody or fragment of any preceding aspect, wherein the    human is suffering from or at risk of an inflammatory bowel disease    (IBD), allogeneic transplant rejection, graft-versus-host disease    (GvHD), uveitis, pyoderma gangrenosum, giant cell arteritis,    Schnitzler syndrome, non-infectious scleritis, diabetes or airway    inflammation and said method treats or prevents IBD, allogeneic    transplant rejection, GvHD, uveitis, pyoderma gangrenosum, giant    cell arteritis, Schnitzler syndrome, non-infectious scleritis,    diabetes or airway inflammation in the human.

In an example of any preceding aspect the human is suffering from or atrisk of an inflammatory or autoimmune disease or condition or has beendiagnosed as such.

In an example, the autoimmune disease or condition is selected from thefollowing:—

-   -   Acute disseminated encephalomyelitis (ADEM)    -   Addison's disease    -   Allergic granulomatosis and angiitis or Churg-Strauss syndrome        (CSS)    -   Alopecia or Alopecia Areata (AA)    -   Anklosing spondylitis    -   Autoimmune chronic active hepatitis (CAH)    -   Autoimmune hemolytic anemia    -   Autoimmune pancreatitis (AIP)    -   Autoimmune retinopathy (AR) see Retinopathy    -   Autoimmunc thrombocytopenic purpura    -   Autoimmune neutropenia    -   Autoimmune Inner Ear Disease (AIED)    -   Antiphospholipid Syndrome (APS)    -   Autoimmune Lymphoproliferative Syndrome (ALPS)    -   Behcet's syndrome    -   Bullus pemphigoid    -   Celiac disease    -   Churg-Strauss Syndrome (CSS) or Allergic Granulomatosis Angiitis    -   Chronic bullous disease of childhood    -   Chronic inflammatory demyelinating Polyradiculoneuropathy (CIDP)    -   Cictricial pemphigoid (CP)    -   Central Nervous System Vasculitis    -   Crohn's Disease    -   Cryoglobulinemia    -   Dermatitis herpetiformis (DH)    -   Discoid lupus erythematosus (DLE)    -   Encephalomyelitis    -   Epidermolysis bullosa acquisita (EBA)    -   Giant Cell Artcritis sec Temporal arteritis    -   Graft-versus-host disease    -   Graves' Disease    -   Gullain-Barre syndrome    -   Hanot Syndrome see Primary biliary Cirrhosis    -   Hashimoto's thyroiditis also called autoimmune thyroiditis and        chronic lymphocytic thyroiditis    -   Hypersensitivity Vasculitis (HV) or small vessel vasculitis    -   Immune-mediated infertility    -   Inflammatory bowel disease    -   Insulin-dependent diabetes mellitus    -   Isolated vasculitis of the Central nervous system or CNS        Vasculitis    -   Isaacs' Syndrome: Neuromyotonia    -   Kawasaki disease (KD)    -   Lambert-Eaton myasthenic syndrome (LEMS)    -   Linear IgA disease    -   Lupus—see Systemic lupus erythematosus    -   Meniere's Disease    -   Microscopic Polyangiitis (MPA)    -   Mixed connective tissue disease or MCTD    -   Monoclonal Gammopathy    -   Myasthenia Gravis    -   Multiple Sclerosis    -   Multifocal motor neuropathy    -   Neuromyotonia or Isaac's syndrome    -   Neutropenia see Autoimmune Neutropenia    -   Oophoritis    -   Opsoclonus-myoclonus syndrome    -   orchitis    -   Paraneoplastic neurologic disorders    -   Pemphigus vulgaris    -   Pemphigus follaceus PF)    -   Pemphigoid gestationis (PG)    -   Pernicious anemia    -   Paraneoplastic pemphigus (PNP)    -   Polyangiitis—see Microscopic polyangiitis    -   Polyarteritis nodosa (PAN)    -   Polymyositis/Dermatomyositis    -   Polymyalgia Rheumatica    -   Primary biliary Cirrhosis (PBC) also called Hanot Syndrome    -   Primary sclerosing cholangitis (PSC)    -   Raynaud's phenomenon    -   Recoverin-associated retinopathy (RAR) see Retinopathy    -   Reactive Arthritis formerly known as Reiter's syndrome,    -   Retinopathy    -   Rheumatoid arthritis (RA)    -   Sarcoidosis    -   Sclerosing cholangitis see Primary Sclerosing Cholangitis    -   Sjogren's syndrome    -   Systemic necrotizing vascolitides    -   Stiff man syndrome or Moersch-Woltmann syndrome    -   Systemic lupus erythematosus    -   Systemic sclerosis (scleroderma)    -   Temporal arteritis or giant cell arteritis (GCV)    -   Takayasu's arteritis    -   Thromboangiitis obliterans or Buerger's disease    -   Thyroiditis with hypothyroidism    -   Thyroiditis with hyperthyroidism    -   Type 1 autoimmunc polyglandular syndrome (PAS)    -   Type II autoimmune polyglandular syndrome    -   Vasculitis    -   Wegener's granulomatosis

In an example of any aspect, configuration, concept or embodiment, thehuman is suffering from uveitis. For example, the uveitis isnon-infectious and/or autoimmune in nature, i.e. is non-infectiousuveitis or is autoimmune uveitis. For example, thenon-infectious/autoimmune uveitis is caused by and/or is associated withBehçet disease, Fuchs heterochromic iridocyclitis, granulomatosis withpolyangiitis, HLA-B27 related uveitis, juvenile idiopathic arthritis,sarcoidosis, spondyloarthritis, sympathetic ophthalmia,tubulointerstitial nephritis or uveitis syndrome. In an example, theuveitis is systemic in nature, i.e. is systemic uveitis. For example,the systemic uveitis is caused by and/or is associated with ankylosingspondylitis, Behçet's disease, chronic granulomatous disease,enthesitis, inflammatory bowel disease, juvenile rheumatoid arthritis,Kawasaki's disease, multiple sclerosis, polyarteritis nodosa, psoriaticarthritis, reactive arthritis, sarcoidosis, systemic lupuserythematosus, Vogt-Koyanagi-Harada syndrome or Whipple's disease.

In an example of any aspect, configuration, concept or embodiment, thehuman is suffering from pyoderma gangrenosum, giant cell arteritis,Schnitzler syndrome or non-infectious scleritis. In an example, thehuman is suffering from pyoderma gangrenosum. In an example, the humanis suffering from giant cell arteritis. In an example, the human issuffering from Schnitzler syndrome. In an example, the human issuffering from non-infectious scleritis.

In an example of any aspect, configuration, concept or embodiment, thehuman is suffering from a hOX40L mediated disease or condition selectedfrom an autoimmune disease or condition, a systemic inflammatory diseaseor condition, or transplant rejection; for example inflammatory boweldisease (IBD), Crohn's disease, rheumatoid arthritis, transplantrejection, allogeneic transplant rejection, graft-versus-host disease(GvHD), ulcerative colitis, systemic lupus erythematosus (SLE),diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity,multiple sclerosis and atherosclerosis, in particular GvHD. In anotherembodiment, the human is suffering from or is at risk from multivisceralorgan transplant rejection.

-   13. An antibody or a fragment thereof, that specifically binds to    hOX40L and competes for binding to said hOX40L with an antibody    selected from the group consisting of 02D10, 10A07, 09H04 and 19H01.

In an example of any aspect, configuration, concept or embodiment,competition is determined by surface plasmon resonance (SPR), suchtechniques being readily apparent to the skilled person. SPR can becarried out using Biacore™, Proteon™ or another standard SPR technique.Such competition may be due, for example, to the antibodies/fragmentsbinding to identical or overlapping epitopes of hOX40L. In an example ofany aspect, configuration, concept or embodiment, competition isdetermined by ELISA, such techniques being readily apparent to theskilled person. In an example of any aspect, configuration, concept orembodiment, competition is determined by homogenous time resolvedfluorescence (HTRF), such techniques being readily apparent to theskilled person. In an example of any aspect, configuration, concept orembodiment, competition is determined by fluorescence activated cellsorting (FACS), such techniques being readily apparent to the skilledperson. In one aspect, the HTRF, ELISA and/or FACS methods are carriedout as described in the Examples hereinbelow.

-   14. The antibody or fragment of aspect 13, wherein the antibody or    fragment is according to any one of aspects 1 to 12.-   15. The antibody or fragment of any preceding aspect, comprising    lambda light chain variable domains (optionally which are human).

In an example of any aspect, configuration, concept or embodiment of thepresent invention, the variable domains of the antibody or fragment arehuman or humanised. Additionally, optionally the antibody or fragmentfurther comprises human or humanised constant regions (e.g., human Fcand/or human CL). In an example of any aspect of the present invention,the variable domains of the antibody or fragment are produced by atransgenic animal (e.g., a rodent, mouse, rat, rabbit, chicken, sheep,Camelid or shark). In an example of any aspect of the present invention,the variable domains of the antibody or fragment are produced oridentified by phage display, ribosome display or yeast display.

In an example of any aspect, configuration, concept or embodiment of thepresent invention, the antibody or fragment is recombinant.

In an example of any aspect, configuration, concept or embodiment of thepresent invention, the antibody or fragment is produced by a recombinantmammalian, bacterial, insect, plant or yeast cell. In an example, themammalian cell is a CHO or HEK293 cell and the antibody or fragmentcomprises CHO or HEK293 cell glycosylation.

In an example of any aspect, configuration, concept or embodiment of thepresent invention, the antibody or fragment is isolated.

-   16. The antibody or fragment of any preceding aspect, comprising a    VH domain which comprises a HCDR1 sequence selected from the group    consisting of the HCDR1 of:    -   a. 02D10, and wherein the antibody or fragment competes with        02D10 for binding to said hOX40L;    -   b. 10A07, and wherein the antibody or fragment competes with        10A07 for binding to said hOX40L;    -   c. 09H04, and wherein the antibody or fragment competes with        09H04 for binding to said hOX40L; and    -   d. 19H01, and wherein the antibody or fragment competes with        19H01 for binding to said hOX40L-   17. The antibody or fragment of any preceding aspect, comprising a    VH domain which comprises a HCDR2 sequence selected from the group    consisting of the HCDR2 of:    -   a. 02D10, and wherein the antibody or fragment competes with        02D10 for binding to said hOX40L;    -   b. 10A07, and wherein the antibody or fragment competes with        10A07 for binding to said hOX40L;    -   c. 09H04, and wherein the antibody or fragment competes with        09H04 for binding to said hOX40L; and    -   d. 19H01, and wherein the antibody or fragment competes with        19H01 for binding to said hOX40L-   18. The antibody or fragment of any preceding aspect, comprising a    VH domain which comprises a HCDR3 sequence selected from the group    consisting of the HCDR3 of:    -   a. 02D10, and wherein the antibody or fragment competes with        02D10 for binding to said hOX40L;    -   b. 10A07, and wherein the antibody or fragment competes with        10A07 for binding to said hOX40L;    -   c. 09H04, and wherein the antibody or fragment competes with        09H04 for binding to said hOX40L; and    -   d. 19H01, and wherein the antibody or fragment competes with        19H01 for binding to said hOX40L-   19. The antibody or fragment of any preceding aspect, comprising a    VH domain which comprises (i) the CDR1 and 2, (ii) CDR1 and 3, (iii)    CDR2 and 3 or (iv) CDR1, 2 and 3 sequences:    -   a. recited in (a) of aspects 16-18, and wherein the antibody or        fragment competes with 02D10 for binding to said hOX40L;    -   b. recited in (b) of aspects 16-18, and wherein the antibody or        fragment competes with 10A07 for binding to said hOX40L;    -   c. recited in (c) of aspects 16-18, and wherein the antibody or        fragment competes with 09H04 for binding to said hOX40L; or    -   d. recited in (d) of aspects 16-18, and wherein the antibody or        fragment competes with 19H01 for binding to said hOX40L.-   20. The antibody or fragment of any preceding aspect, comprising a    VH domain which comprises an amino acid sequence selected from the    group consisting of the VH amino acid sequences in the sequence    listing.

In an aspect, the invention provides an anti-hOX40L antibody or fragment(optionally according to any other aspect recited herein) comprising aVH domain which comprises an amino acid sequence selected from the groupconsisting of the VH amino acid sequences in the sequence listing. In anaspect, the VH domain comprises an amino acid sequence selected from SeqID No:2, Seq ID No:34, Seq ID No:66, Seq ID No:94, Seq ID No:122, Seq IDNo:124, Seq ID NO:126, Seq ID No:128, Seq ID No:132 or Seq ID No:134.

In another example of the invention, the antibody or fragment comprisesa VH domain amino acid sequence set out in the sequence listing below.Additionally or alternatively, the antibody or fragment comprises aHCDR1 domain amino acid sequence set out in the sequence listing below(i.e. Seq ID No:4, Seq ID No:10, Seq ID No:36, Seq ID No:42, Seq IDNo:68, Seq ID No:74, Seq ID No:96 or Seq ID No:102, in particular, SeqID No:36 or Seq ID No:42). Additionally or alternatively, the antibodyor fragment comprises a HCDR2 domain amino acid sequence set out in thesequence listing below (i.e. Seq ID No:6, Seq ID No:12, Seq ID No:38,Seq ID No:44, Seq ID No:70, Seq ID No:76, Seq ID No:98 or Seq ID No:104,in particular Seq ID No:38 or Seq ID No:44). Additionally oralternatively, the antibody or fragment comprises a HCDR3 domain aminoacid sequence set out in the sequence listing below (i.e. Seq ID No:8,Seq ID No:14, Seq ID No:40, Seq ID No:46, Seq ID No:72, Seq ID No:78,Seq ID No:100 or Seq ID No:106, in particular Seq ID No:40 or Seq IDNo:46).

In an example of the invention, the antibody or fragment comprises a VLdomain amino acid sequence set out in the sequence listing below.Additionally or alternatively, the antibody or fragment comprises aLCDR1 domain amino acid sequence set out in the sequence listing below(i.e. Seq ID No:18, Seq ID No:24, Seq ID No:50, Seq ID No:56, Seq IDNo:82, Seq ID No:88, Seq ID No:110 or Seq ID No:116, in particular SeqID No:50 or Seq ID No:56). Additionally or alternatively, the antibodyor fragment comprises a LCDR2 domain amino acid sequence set out in thesequence listing below (i.e. Seq ID No:20, Seq ID No:26, Seq ID No:52,Seq ID No:58, Seq ID No:84, Seq ID No:90, Seq ID No:112 or Seq IDNo:118, in particular Seq ID No:52 or Seq ID No:58). Additionally oralternatively, the antibody or fragment comprises a LCDR3 domain aminoacid sequence set out in the sequence listing below (i.e. Seq ID No:22,Seq ID No:28, Seq ID No:54, Seq ID No:60, Seq ID No:86, Seq ID No:92,Seq ID No:114 or Seq ID No:120, in particular Seq ID No:54 or Seq IDNo:60).

In an example of any aspect herein, the antibody or fragment comprises aheavy chain comprising a constant region selected from the groupconsisting of the heavy chain constant region SEQ ID NOs in the sequencelisting (i.e. any of Seq ID Nos: 126, 128, 132, or 134, in particularthe constant region of Seq ID No:128); and optionally a VH domain asrecited in aspect 19 or 20. In an example, the antibody or fragmentcomprises two copies of such a heavy chain. In another example, theheavy chain comprise a rodent, rat, mouse, human, rabbit, chicken,Camelid, sheep, bovine, non-human primate or shark constant region(e.g., Fc), in particular a mouse constant region.

In an example of any aspect herein, the antibody or fragment comprises aheavy chain comprising a gamma (e.g., human gamma) constant region,e.g., a human gamma′ constant region. In another example of any aspectherein, the antibody of fragment comprises a human gamma 4 constantregion. In another embodiment, the heavy chain constant region does notbind Fc-γ receptors, and e.g. comprises a Leu235Glu mutation (i.e. wherethe wild type leucine residue is mutated to a glutamic acid residue). Inanother embodiment, the heavy chain constant region comprises aSer228Pro mutation to increase stability. In another embodiment, theheavy chain constant region is IgG4 comprising both the Leu235Glumutation and the Ser228Pro mutation. This heavy chain constant region isreferred to as “IgG4-PE” herein.

In an example of any aspect herein, the antibody or fragment ischimaeric, e.g., it comprises human variable domains and non-human(e.g., rodent, mouse or rat, such as mouse) constant regions.

-   21. The antibody or fragment of any one of aspects 16 to 20,    comprising first and second copies of said VH domain.-   22. The antibody or fragment of any preceding aspect, comprising a    VL domain which comprises a LCDR1 sequence selected from the group    consisting of the LCDR1 of:    -   a. 02D10, and wherein the antibody or fragment competes with        02D10 for binding to said hOX40L;    -   b. 10A07, and wherein the antibody or fragment competes with        10A07 for binding to said hOX40L;    -   c. 09H04, and wherein the antibody or fragment competes with        09H04 for binding to said hOX40L; and    -   d. 19H01, and wherein the antibody or fragment competes with        19H01 for binding to said hOX40L-   23. The antibody or fragment of any preceding aspect, comprising a    VL domain which comprises a LCDR2 sequence selected from the group    consisting of the LCDR2 of:    -   a. 02D10, and wherein the antibody or fragment competes with        02D10 for binding to said hOX40L;    -   b. 10A07, and wherein the antibody or fragment competes with        10A07 for binding to said hOX40L;    -   c. 09H04, and wherein the antibody or fragment competes with        09H04 for binding to said hOX40L; and    -   d. 19H01, and wherein the antibody or fragment competes with        19H01 for binding to said hOX40L-   24. The antibody or fragment of any preceding aspect, comprising a    VL domain which comprises a LCDR3 sequence selected from the group    consisting of the LCDR3 of:    -   a. 02D10, and wherein the antibody or fragment competes with        02D10 for binding to said hOX40L;    -   b. 10A07, and wherein the antibody or fragment competes with        10A07 for binding to said hOX40L;    -   c. 09H04, and wherein the antibody or fragment competes with        09H04 for binding to said hOX40L; and    -   d. 19H01, and wherein the antibody or fragment competes with        19H01 for binding to said hOX40L-   25. The antibody or fragment of any preceding aspect, comprising a    VL domain which comprises (i) the CDR1 and 2, (ii) CDR1 and 3, (iii)    CDR2 and 3 or (iv) CDR1, 2 and 3 sequences:    -   a. recited in (a) of aspects 22-24, and wherein the antibody or        fragment competes with 02D10 for binding to said hOX40L;    -   b. recited in (b) of aspects 22-24, and wherein the antibody or        fragment competes with 10A07 for binding to said hOX40L;    -   c. recited in (c) of aspects 22-24, and wherein the antibody or        fragment competes with 09H04 for binding to said hOX40L; or    -   d. recited in (d) of aspects 22-24, and wherein the antibody or        fragment competes with 19H01 for binding to said hOX40L.-   26. The antibody or fragment of any preceding aspect, comprising a    VL domain which comprises an amino acid sequence selected from the    group consisting of the VL amino acid sequences in the sequence    listing.

In an aspect of the invention, there is provided an anti-hOX40L antibodyor fragment (optionally according to any other aspect herein),comprising a VL domain which comprises an amino acid sequence selectedfrom the group consisting of the VL amino acid sequences in the sequencelisting (i.e. Seq ID No:16, Seq ID No:48, Seq ID No:80 or Seq ID No:108,in particular Seq ID No:48).

In an example of any aspect herein, the antibody or fragment comprises alight chain (e.g., lambda light chain) comprising a constant regionselected from the group consisting of the light chain constant regionsequences in the sequence listing (i.e. Seq ID No:136, Seq ID No:138,Seq ID No:140, Seq ID No:142, Seq ID No:144, Seq ID No:146, Seq IDNo:148, Seq ID No:152, Seq ID No:154, Seq ID No:156, Seq ID No:158, SeqID No:160, Seq ID No:162, Seq ID No:164 or Seq ID No:166); andoptionally a VL domain (e.g., lambda VL) as recited in aspect 25 or 26.In an example, the antibody or fragment comprises two copies of such alight chain (optionally also two copies of the heavy chain describedabove). In another example, the light chain comprise a rodent, rat,mouse, human, rabbit, chicken, Camelid, sheep, bovine, non-human primateor shark constant region.

In an example of any aspect herein, the antibody or fragment comprises alight chain (e.g., kappa light chain) comprising a constant regionselected from the group consisting of the light chain constant regionsequences in the sequence listing (i.e. Seq ID No:136, Seq ID No:138,Seq ID No:140, Seq ID No:142, Seq ID No:144, Seq ID No:146, Seq IDNo:148, Seq ID No:152, Seq ID No:154, Seq ID No:156, Seq ID No:158, SeqID No:160, Seq ID No:162, Seq ID No:164 or Seq ID No: 166); andoptionally a VL domain (e.g., kappa VL) as recited in aspect 25 or 26.In an example, the antibody or fragment comprises two copies of such alight chain (optionally also two copies of the heavy chain describedabove). In another example, the light chain comprise a rodent, rat,mouse, human, rabbit, chicken, Camelid, sheep, bovine, non-human primateor shark constant region.

In an example, the antibody or fragment comprises a lambda light chaincomprising a constant region selected from the group consisting of thelight chain constant region sequences in the sequence listing (i.e. SeqID No:146, Seq ID No:148, Seq ID No:152, Seq ID No:154, Seq ID No:156,Seq ID No:158, Seq ID No:160, Seq ID No:162, Seq ID No:164 or Seq IDNo:166); and optionally a lambda VL domain.

In an example, the antibody or fragment comprises a kappa light chaincomprising a constant region selected from the group consisting of thelight chain constant region sequences in the sequence listing (i.e. i.e.Seq ID No:136, Seq ID No:138, Seq ID No:140, Seq ID No:142 or Seq IDNo:144); and optionally a kappa VL domain.

In an example, the VL domains of the antibody or fragment are lambdaLight chain variable domains. In an example, the VL domains of theantibody or fragment are kappa Light chain variable domains.

-   27. The antibody or fragment of any one of aspects 22 to 26,    comprising first and second copies of said VL domain.-   28. The antibody or fragment of any preceding aspect, wherein the    hOX40L is human cell surface-expressed hOX40L, e.g., on endothelial    cells (e.g., an airway or GI tract endothelial cell).

In another embodiment, the epithelial cells comprise cells selected fromthe group consisting of gastrointestinal cells, colon cells, intestinalcells, ocular cells and airway (e.g., lung) epithelial cells. In anotherembodiment, the epithelial cells comprise cells selected from the groupconsisting of gastrointestinal cells, colon cells, intestinal cells andocular cells. In a further embodiment, the epithelial cells compriseocular cells.

-   29. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases the proliferation of human PBMCs or    T-cells in the presence of hOX40L in an in vitro mixed lymphocyte    reaction (MLR) assay by at least 20, 30, 40, 50 or 60% compared to    the proliferation of human PBMCs or T-cells in the presence of    hOX40L in an in vitro control MLR assay in the absence of an    antibody that is specific for hOX40L. An illustration of a suitable    assay is provided in the examples below.-   30. The antibody or fragment of aspect 29, wherein the hOX40L in the    assay is surface-expressed on human dendritic cells (DC cells).

An illustration of a suitable assay is provided in the examples below.

-   31. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases NF-κB activity in human HT-1080 cells    expressing hOX40 receptor in vitro in the presence of hOX40L.

In an example, the antibody or fragment the decrease in NF-κB activityis determined by detecting a decrease in IL-8 secretion by HT-1080 cells(ATCC® CCL-121) (optionally transfected with hOX40 Receptor, in thepresence of hOX40) in vitro.

-   32. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases IL-8 secretion from human HT-1080    cells expressing hOX40 receptor in vitro in the presence of hOX40L.-   33. The antibody or fragment of aspect 32, wherein the antibody or    fragment decreases IL-8 secretion by at least 20, 30, 40, 50 or 60%    compared to the IL-8 production by HT-1080 cells expressing hOX40    receptor in vitro in the presence of hOX40L in the absence of an    antibody that is specific for hOX40L.-   34. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases hOX40L-stimulated human T-cell    proliferation in vitro.-   35. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases hOX40L-stimulated IL-2 secretion from    human T-cells in vitro.-   36. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases cytokine secretion mediated by the    interaction of human dendritic cells (DC cells) with human T-cells,    wherein the cytokine is selected from one, two, more or all of TNF    alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13,    IL-17, RANTES and interferon gamma.

This can be assessed, for example, using a MLR in vitro assay (e.g., aDC/T-cell MLR in vitro assay). An illustration of a suitable assay isprovided in the examples below.

In an example, the DC cells are mismatched to the T-cells, e.g., MHCmis-matched, as is possible for example when the DC cells are from ahuman that is different from the T-cell human source. In an example, theDC cells are produced by in vitro induction of human monocytes withGMCSF and IL-4.

-   37. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases interferon gamma secretion by at    least 20, 30, 40, 50 or 60% compared to the production of interferon    gamma mediated by the interaction of human dendritic cells (DC    cells) with human T-cells in the absence of an antibody that is    specific for hOX40L.-   38. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases TNF alpha secretion by at least 20,    30, 40, 50 or 60% compared to the production of TNF alpha mediated    by the interaction of human dendritic cells (DC cells) with human    T-cells in the absence of an antibody that is specific for hOX40L.-   39. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases IL-2 secretion by at least 10, 20,    30, 40, 50 or 60% compared to the production of IL-2 mediated by the    interaction of human dendritic cells (DC cells) with human T-cells    in the absence of an antibody that is specific for hOX40L.-   40. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases cytokine secretion (e.g., leukocyte    cytokine secretion) in a human peripheral blood mononuclear cell    (PBMC) mixed lymphocyte (MLR) assay, wherein the cytokine is    selected from one, two, more or all of TNF alpha, IL-2, IL-4, IL-3,    IL-6, IL-8, IL-10, IL-17, RANTES and interferon gamma.-   41. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases interferon gamma secretion by at    least 20, 30, 40, 50 or 60% compared to the production of interferon    gamma in a human PBMC MLR assay in the absence of an antibody that    is specific for hOX40L.

In one embodiment, the comparison is to the production of interferongamma in a human PBMC MLR assay in the absence of antibody.

-   42. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases TNF alpha secretion by at least 20,    30, 40, 50 or 60% compared to the production of TNF alpha in a human    PBMC MLR assay in the absence of an antibody that is specific for    hOX40L.-   43. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment decreases IL-2 secretion by at least 10, 20,    30, 40, 50 or 60% compared to the production of IL-2 in a human PBMC    MLR assay in the absence of an antibody that is specific for hOX40L.-   44. The antibody or fragment of any one of aspects 36 to 43, wherein    the cells are primary cells.

A “primary cell” refers to a cell in a human or such a cell that hasbeen taken from the patient for binding to the antibody or fragment ofthe invention in vitro (as may be useful, for example, in a method ofdiagnosis of OX40L status or disease/condition status in the human).Primary cells as used herein are not cells of human cell lines, whichtypically have undergone many cultures in vitro. The ability of theantibody or fragment of the invention to specifically inhibit hOX40Lbinding to receptor in this embodiment is advantageous since it providesa direct indication of the utility for addressing cells in humanpatients suffering or at risk of a hOX40L-mediated disease or condition.

-   45. The antibody or fragment of any preceding aspect, wherein the    antibody or fragment inhibits binding of hOX40L to a hOX40L receptor    (e.g., hOX40) with an IC₅₀ of 1×10⁻⁸ or less in a HTRF (homogenous    time resolved fluorescence) assay.

In an example, the IC₅₀ is in the range from 1×10⁻⁸ to 1×10⁻¹¹ or in therange from 1×10⁻⁹ to 1×10⁻¹⁰.

-   46. A pharmaceutical composition for treating and/or preventing a    OX40L-mediated condition or disease, the composition comprising an    antibody or fragment of any preceding aspect and a diluent,    excipient or carrier; and optionally further comprising an    anti-inflammatory drug.

In an example, the anti-inflammatory drug is independently selected fromthe group consisting of corticosteroids (e.g. methylprednisolone),anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-VLA4 antibodies(e.g. natalizumab), anti-LFA1 antibodies, anti-complement C5 antibodies(e.g. eculizumab), anti-a4b7 integrin antibodies (e.g. vedolizumab),anti-IL6 antibodies (e.g. tocilizumab), anti-IL2R antibodies (e.g.basilixumab) or anti-TNFa antibodies/TNFa-Fc molecules (e.g. etanercept,adalimumab, infliximab, golimumab, certolizumab pegol). In an example,the anti-inflammatory drug is independently selected from the groupconsisting of corticosteroids (e.g. methylprednisolone) and anti-LFA1antibodies.

-   47. A pharmaceutical composition or kit for treating and/or    preventing a OX40L-mediated condition or disease, the composition or    kit comprising an antibody or fragment of the invention (and    optionally an anti-inflammatory drug) optionally in combination with    a label or instructions for use to treat and/or prevent said disease    or condition in a human; optionally wherein the label or    instructions comprise a marketing authorisation number (e.g., an FDA    or EMA authorisation number); optionally wherein the kit comprises    an IV or injection device that comprises the antibody or fragment.-   48. A nucleic acid that encodes the HCDR3 of an antibody recited in    any one of aspects 1 to 45.

In one embodiment, the HCDRs herein are according to Kabat nomenclature.In another embodiment, the HCDRs herein are according to the IMGTnomenclature.

-   49. The nucleic acid of aspect 48 comprising a nucleotide sequence    that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical or is    100% identical to a HCDR3 sequence in the sequence listing.

In an aspect, the invention provides a nucleic acid comprising anucleotide sequence that encodes a VH domain of an anti-hOX40L antibody,wherein the nucleotide sequence comprises a HCDR3 sequence that is atleast 80, 85, 90, 95, 96, 97, 98 or 99% identical or is 100% identicalto a HCDR3 sequence in the sequence listing. Optionally, the antibody isaccording to any other aspect herein.

In another embodiment, there is provided the nucleic acid of aspect 48comprising a nucleotide sequence that is 100% identical to a HCDR3sequence in the sequence listing, except for 1, 2 or 3 nucleotidesubstitutions, wherein each substitution produces no amino acid changeor produces a conservative amino acid change (i.e., the nucleotidesubstitution is a synonymous substitution) in the corresponding proteinsequence. The skilled person will be familiar with conservative aminoacid changes.

Amino acid substitutions include alterations in which an amino acid isreplaced with a different naturally-occurring amino acid residue. Suchsubstitutions may be classified as “conservative”, in which case anamino acid residue contained in a polypeptide is replaced with anothernaturally occurring amino acid of similar character either in relationto polarity, side chain functionality or size. Such conservativesubstitutions are well known in the art. Substitutions encompassed bythe present invention may also be “non-conservative”, in which an aminoacid residue which is present in a peptide is substituted with an aminoacid having different properties, such as naturally-occurring amino acidfrom a different group (e.g., substituting a charged or hydrophobicamino; acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid.

Additionally or alternatively, there is provided the nucleic acid ofaspect 49 comprising a nucleotide sequence that is 100% identical to aHCDR3 sequence in the sequence listing, except for 1, 2, 3, 4, 5, 6 or 7synonymous nucleotide substitutions and no, 1, 2 or 3 nucleotidesubstitutions that produce conservative amino acid changes in thecorresponding protein sequence.

-   50. A nucleic acid that encodes the HCDR2 of an antibody recited in    any one of aspects 1 to 45; optionally wherein the nucleic acid is    according to aspect 48 or 49.-   51. The nucleic acid of aspect 50 comprising a nucleotide sequence    that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical or is    100% identical to a HCDR2 sequence in the sequence listing.

In an aspect, the invention provides a nucleic acid comprising anucleotide sequence that encodes a VH domain of an anti-hOX40L antibody,wherein the nucleotide sequence comprises a HCDR2 sequence that is atleast 80, 85, 90, 95, 96, 97, 98 or 99% identical or is 100% identicalto a HCDR2 sequence in the sequence listing. Optionally, the antibody isaccording to any other aspect herein.

In another embodiment, there is provided the nucleic acid of aspect 51comprising a nucleotide sequence that is 100% identical to a HCDR2sequence in the sequence listing, except for 1, 2 or 3 nucleotidesubstitutions, wherein each substitution produces no amino acid changeor produces a conservative amino acid change (i.e., the nucleotidesubstitution is a synonymous substitution) in the corresponding proteinsequence. The skilled person will be familiar with conservative aminoacid changes.

Additionally or alternatively, there is provided the nucleic acid ofaspect 50 comprising a nucleotide sequence that is 100% identical to aHCDR2 sequence in the sequence listing, except for 1, 2, 3, 4, 5, 6 or 7synonymous nucleotide substitutions and no, 1, 2 or 3 nucleotidesubstitutions that produce conservative amino acid changes in thecorresponding protein sequence.

-   52. A nucleic acid that encodes the HCDR1 of an antibody recited in    any one of aspects 1 to 45; optionally wherein the nucleic acid is    according to any one of aspects 48 to 51.-   53. The nucleic acid of aspect 52 comprising a nucleotide sequence    that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to or    is 100% identical to a HCDR1 sequence in the sequence listing.

In an aspect, the invention provides a nucleic acid comprising anucleotide sequence that encodes a VH domain of an anti-hOX40L antibody,wherein the nucleotide sequence comprises a HCDR1 sequence that is atleast 80, 85, 90, 95, 96, 97, 98 or 99% identical or is 100% identicalto a HCDR1 sequence in the sequence listing. Optionally, the antibody isaccording to any other aspect herein.

In another embodiment, there is provided the nucleic acid of aspect 52comprising a nucleotide sequence that is 100% identical to a HCDR1sequence in the sequence listing, except for 1, 2 or 3 nucleotidesubstitutions, wherein each substitution produces no amino acid changeor produces a conservative amino acid change (i.e., the nucleotidesubstitution is a synonymous substitution) in the corresponding proteinsequence. The skilled person will be familiar with conservative aminoacid changes.

Additionally or alternatively, there is provided the nucleic acid ofaspect 52 comprising a nucleotide sequence that is 100% identical to aHCDR1 sequence in the sequence listing, except for 1, 2, 3, 4, 5, 6 or 7synonymous nucleotide substitutions and no, 1, 2 or 3 nucleotidesubstitutions that produce conservative amino acid changes in thecorresponding protein sequence.

-   54. A nucleic acid that encodes a VH domain and/or a VL domain of an    antibody recited in any one of aspects 1 to 45.-   55. The nucleic acid of aspect 54 comprising a nucleotide sequence    that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to or    is 100% identical to a VH domain nucleotide sequence in the sequence    listing.

In another embodiment, there is provided the nucleic acid of aspect 54comprising a nucleotide sequence that is 100% identical to a VH domainnucleotide sequence in the sequence listing, except for 1, 2 or 3nucleotide substitutions, wherein each substitution produces no aminoacid change or produces a conservative amino acid change (i.e., thenucleotide substitution is a synonymous substitution) in thecorresponding protein sequence. The skilled person will be familiar withconservative amino acid changes.

Additionally or alternatively, there is provided the nucleic acid ofaspect 54 comprising a nucleotide sequence that is 100% identical to aVH domain nucleotide sequence in the sequence listing, except for 1, 2,3, 4, 5, 6 or 7 synonymous nucleotide substitutions and no, 1, 2 or 3nucleotide substitutions that produce conservative amino acid changes inthe corresponding protein sequence.

-   56. The nucleic acid of aspect 54 or 55 comprising a nucleotide    sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99%    identical to or is 100% identical to a VL domain nucleotide sequence    in the sequence listing.

In another embodiment, there is provided the nucleic acid of aspect 54or 55 comprising a nucleotide sequence that is 100% identical to a VLdomain nucleotide sequence in the sequence listing, except for 1, 2 or 3nucleotide substitutions, wherein each substitution produces no aminoacid change or produces a conservative amino acid change (i.e., thenucleotide substitution is a synonymous substitution) in thecorresponding protein sequence. The skilled person will be familiar withconservative amino acid changes.

Additionally or alternatively, there is provided the nucleic acid ofaspect 54 or 55 comprising a nucleotide sequence that is 100% identicalto a VL domain nucleotide sequence in the sequence listing, except for1, 2, 3, 4, 5, 6 or 7 synonymous nucleotide substitutions and no, 1, 2or 3 nucleotide substitutions that produce conservative amino acidchanges in the corresponding protein sequence.

-   57. A nucleic acid that encodes a heavy chain or a light chain of an    antibody recited in any one of aspects 1 to 45.-   58. The nucleic acid of aspect 57, comprising a nucleotide sequence    as recited in any one of aspects 48 to 56.-   59. A vector (e.g., a mammalian expression vector) comprising the    nucleic acid of any one of aspects 48 to 58; optionally wherein the    vector is a CHO or HEK293 vector. In an example, the vector is a    yeast vector, e.g., a Saccharomyces or Pichia vector.-   60. A host comprising the nucleic acid of any one of aspects 48 to    58 or the vector of aspect 59. In an example, the host is a    mammalian (e.g., human, e.g., CHO or HEK293) cell line or a yeast or    bacterial cell line.-   61. Use of an antibody or a fragment thereof, that specifically    binds to hOX40L in the manufacture of a medicament for    administration to a human, for treating or preventing a    hOX40L-mediated disease or condition in the human by decreasing one,    more or all of    -   a. secretion of a cytokine selected from TNF alpha, IL-2, IL-3,        IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES and        interferon gamma in the human;    -   b. the proliferation of leukocytes of the human; and    -   c. binding of hOX40 receptor expressed by human T-cells with        endothelial cell expressed hOX40L.

The features of any of the previous aspects, configurations, concepts,examples or embodiments optionally apply mutatis mutandis to this use.

In an example, the human is suffering from or at risk of asthma and theantibody or fragment is for decreasing IgE in the human, therebytreating, preventing or reducing asthma in the human.

-   62. A method of treating or preventing a hOX40L-mediated disease or    condition in a human by decreasing one, more or all of    -   a. secretion of a cytokine selected from TNF alpha, IL-2, IL-3,        IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES and        interferon gamma in the human;    -   b. the proliferation of leukocytes of the human; and    -   c. binding of hOX40 receptor expressed by human T-cells with        endothelial cell expressed hOX40L;    -   wherein the method comprises administering to said human a        therapeutically effective amount of an antibody or fragment that        specifically binds to hOX40L.

The features of any of the previous aspects, examples or embodimentsoptionally apply mutatis mutandis to this method.

The method of the invention treats or prevents said disease or conditionin the human. A “therapeutically effective amount” of the antibody orfragment is that amount (administered in one or several doses, which maybe spaced in time, e.g., substantially monthly administration) that iseffective to bring about said treatment or prevention. This will bereadily apparent to the skilled person and may vary according to theparticular human patient and disease or condition being addressed.

In an example, the human is suffering from or at risk of asthma and theantibody or fragment decreases IgE in the human, thereby treating,preventing or reducing asthma in the human.

-   63. The method or use of aspect 61 or 62, for treating or preventing    said hOX40L-mediated disease, condition or epithelial cell damage in    said human by decreasing the proliferation of T-cells in said human.-   64. The method or use of any one of aspects 61 to 63, for treating    or preventing said hOX40L-mediated disease, condition or epithelial    cell damage in said human by antagonising the interaction between    hOX40L and leukocytes of the human, wherein the proliferation of    leukocytes is decreased.-   65. The method or use of any one of aspects 61 to 64, for treating    or preventing said hOX40L-mediated disease, condition or epithelial    cell damage in said human by decreasing the proliferation of    leukocytes of the human by antagonising the OX40L/OX40L receptor    interaction mediated by T-cells in said human.-   66. The method or use of any one of aspects 61 to 65, for treating    or preventing said hOX40L-mediated disease, condition or epithelial    cell damage in said human by decreasing the secretion of IL-8    cytokine in the human.-   67. The method of aspect 66, for treating or preventing said    disease, condition or epithelial cell damage by decreasing the    secretion of said IL-8 mediated by the interaction of dendritic    cells (DC cells) with T-cells in the human.-   68. The method or use of any one of aspects 61 to 67, wherein    gastrointestinal cell, colon cell, intestinal cell or airway (e.g.,    lung) cell damage is a symptom or cause of said disease or condition    in humans.

In another embodiment, the epithelial cells comprise cells selected fromthe group consisting of gastrointestinal cells, colon cells, intestinalcells, ocular cells and airway (e.g., lung) epithelial cells. In anotherembodiment, the epithelial cells comprise cells selected from the groupconsisting of gastrointestinal cells, colon cells, intestinal cells andocular cells. In a further embodiment, the epithelial cells compriseocular cells.

-   69. The method or use of any one of aspects 61 to 68, wherein the    human is suffering from or at risk of an inflammatory bowel disease    (TBD), allogeneic transplant rejection, graft-versus-host disease    (GvHD), diabetes or airway inflammation and said method treats or    prevents IBD, allogeneic transplant rejection, GvHD, diabetes or    airway inflammation in the human.-   69a. The method or use of any one of aspects 61 to 68, wherein the    human is suffering from or at risk of an inflammatory bowel disease    (IBD), allogeneic transplant rejection, graft-versus-host disease    (GvHD), uveitis, pyoderma gangrenosum, giant cell arteritis,    Schnitzler syndrome, non-infectious scleritis, diabetes or airway    inflammation and said method treats or prevents IBD, allogeneic    transplant rejection, GvHD, uveitis, pyoderma gangrenosum, giant    cell arteritis, Schnitzler syndrome, non-infectious scleritis,    diabetes or airway inflammation in the human.

In any aspect, configuration, concept or embodiment, the human issuffering from or at risk of a hOX40L-mediated disease or conditionselected from an autoimmune disease or condition, a systemicinflammatory disease or condition, or transplant rejection; for exampleinflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis,transplant rejection, allogeneic transplant rejection, graft-versus-hostdisease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE),diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity,multiple sclerosis and atherosclerosis, in particular GvHD.

-   70. The method or use of any one of aspects 61 to 69a, wherein the    antibody or fragment is according to any one of aspects 1 to 45 or    any example, configuration, concept, aspect or embodiment described    herein.-   71. The antibody, fragment, composition, kit, method or use of any    preceding aspect, for treating or preventing an inflammatory or    automimmune disease or condition in a human or for reducing or    preventing angiogenesis in a human.-   72. The antibody, fragment, composition, kit, method or use of any    preceding aspect, wherein the disease or condition is selected from    the group consisting of an inflammatory bowel disease (IBD), Crohn's    disease, rheumatoid arthritis, psoriasis, bronchiolitis, gingivitis,    transplant rejection, allogeneic transplant rejection,    graft-versus-host disease (GvHD), asthma, adult respiratory distress    syndrome (ARDS), septic shock, ulcerative colitis, Sjorgen's    syndrome, airway inflammation, systemic lupus erythematosus (SLE),    diabetes, contact hypersensitivity, multiple sclerosis and    atherosclerosis.-   72a. The antibody, fragment, composition, kit, method or use of any    preceding aspect, wherein the disease or condition is selected from    the group consisting of an inflammatory bowel disease (IBD), Crohn's    disease, rheumatoid arthritis, psoriasis, bronchiolitis, gingivitis,    transplant rejection, allogeneic transplant rejection,    graft-versus-host disease (GvHD), asthma, adult respiratory distress    syndrome (ARDS), septic shock, ulcerative colitis, Sjorgen's    syndrome, airway inflammation, systemic lupus erythematosus (SLE),    uveitis, pyoderma gangrenosum, giant cell arteritis, Schnitzler    syndrome, non-infectious scleritis, diabetes, contact    hypersensitivity, multiple sclerosis and atherosclerosis.

In any aspect, configuration, concept or embodiment, the human issuffering from or at risk of a hOX40L-mediated disease or conditionselected from an autoimmunc disease or condition, a systemicinflammatory disease or condition, or transplant rejection; for exampleinflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis,transplant rejection, allogeneic transplant rejection, graft-versus-hostdisease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE),diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity,multiple sclerosis and atherosclerosis, in particular GvHD.

In an example, the disease or condition is an OX40L-mediated disease orcondition disclosed in U.S. Pat. No. 7,812,133 or EP1791869.

In an example, the disease or condition is an inflammatory or autoimmunedisease or condition. In an example, the disease or condition istransplant rejection.

As used herein, inflammatory disease or condition refers to pathologicalstates resulting in inflammation, for example caused by neutrophilchemotaxis. Examples of such disorders include inflammatory skindiseases including psoriasis; responses associated with inflammatorybowel disease (such as Crohn's disease and ulcerative colitis); ischemicreperfusion; adult respiratory distress syndrome; dermatitis;meningitis; encephalitis; uveitis; autoimmune diseases such asrheumatoid arthritis, Sjorgen's syndrome, vasculitis; diseases involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder, multiple organ injury syndrome secondary to septicaemia ortrauma; alcoholic hepatitis, bacterial pneumonia, antigen-antibodycomplex mediated diseases; inflammations of the lung, includingpleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis,bronchiectasis, and cystic fibrosis; etc. The preferred indications arebacterial pneumonia and inflammatory bowel disease such as ulcerativecolitis. The invention is thus in an example provided for treating orpreventing any one or more of such conditions.

In an example, the disease or condition is cancer.

In an example, the disease is uveitis, such as systemic uveitis orautoimmune/non-infectious uveitis.

-   73. An antibody or a fragment thereof, that specifically binds to    hOX40L and competes for binding to said hOX40L with the antibody    02D10, wherein the antibody or fragment comprises a VH domain which    comprises a HCDR3 comprising the motif VRGXYYY, wherein X is any    amino acid.

The features of the antibodies of any of the aspects, configurations,concepts, examples or embodiments described herein optionally applymutatis mutandis to these antibodies, e.g the antibody may be a humanantibody or chimeric antibody having functional features as describedherein. Competition may be determined as described in any aspect,embodiment, example, concept or configuration described herein, e.g. asdetermined by SPR, ELISA, HTRF or FACS.

In one embodiment, the antibody or fragment competes with the variableregions of 02D10 (e.g. competes with an antibody comprising the heavychain variable region of SEQ ID No: 34 and the light chain variableregion of SEQ ID No:48). In another embodiment, the antibody or fragmentcompetes with 02D10 IgG4-PE having a heavy chain amino acid sequence ofSEQ ID No:62 and a light chain amino acid sequence of SEQ ID No:64.

In another embodiment, the antibody or fragment additionally oralternatively competes with 10A7. In one embodiment, the antibody orfragment competes with the variable regions of 10A7 (e.g. competes withan antibody comprising the heavy chain variable region of SEQ ID No: 2and the light chain variable region of SEQ ID No: 16). In anotherembodiment, the antibody or fragment competes with 02D10 IgG4-PE havinga heavy chain amino acid sequence of SEQ ID No:30 and a light chainamino acid sequence of SEQ ID No:32.

In one embodiment, the amino acid is any naturally-occurring amino acid.

-   74. The antibody or fragment according to aspect 73, where X is a    neutral amino acid, optionally P or G.

In an embodiment, X is P or G. In an embodiment, X is selected from P,N, A or G. In another embodiment, X is selected from P, G or N. Inanother embodiment, X is selected from P, G or A.

-   75. An antibody or a fragment thereof, optionally according to    aspect 73 or 74, that specifically binds to hOX40L and competes for    binding to said hOX40L with the antibody 02D10, wherein the antibody    or fragment comprises a VH domain which comprises the HCDR3 sequence    of SEQ ID NO:40 or 46 or the HCDR3 sequence of SEQ ID NO:40 or 46    comprising less than 5 amino acid substitutions.

The features of the antibodies of any of the aspects, configurations,concepts, examples or embodiments described herein optionally applymutatis mutandis to these antibodies, e.g the antibody may be a humanantibody or chimeric antibody having functional features as describedherein. Competition may be determined as described in any aspect,embodiment, concept, example or configuration described herein, e.g. asdetermined by SPR, ELISA, HTRF or FACS.

In an embodiment, the HCDR3 sequence of SEQ ID NO:40 or 46 comprisesless than 4 amino acid substitutions (i.e. 3 or fewer). In anembodiment, the HCDR3 sequence of SEQ ID NO:40 or 46 comprises less than3 amino acid substitutions (i.e. 2 or 1 substitutions). In anembodiment, the HCDR3 sequence of SEQ ID NO:40 or 46 comprises less than2 amino acid substitutions (i.e. one substitution).

In one embodiment, the antibody or fragment competes with the variableregions of 02D10 (e.g. competes with an antibody comprising the heavychain variable region of SEQ ID No: 34 and the light chain variableregion of SEQ ID No:48). In another embodiment, the antibody or fragmentcompetes with 02D10 IgG4-PE having a heavy chain amino acid sequence ofSEQ ID No:62 and a light chain amino acid sequence of SEQ ID No:64.

In another embodiment, the antibody or fragment additionally oralternatively competes with 10A7. In one embodiment, the antibody orfragment competes with the variable regions of 10A7 (e.g. competes withan antibody comprising the heavy chain variable region of SEQ ID No: 2and the light chain variable region of SEQ ID No:16). In anotherembodiment, the antibody or fragment competes with 02D10 IgG4-PE havinga heavy chain amino acid sequence of SEQ ID No:30 and a light chainamino acid sequence of SEQ ID No:32.

-   76. An antibody or fragment according to any one of aspects 73 to    75, the VH domain comprising a HCDR3 of from 16 to 27 amino acids    and which is derived from the recombination of a human VH gene    segment, a human D gene segment and a human JH gene segment, wherein    the human JH gene segment is IGHJ6 (e.g. IGHJ6*02).

In an embodiment, the human JH gene segment is selected from IGHJ6*01,IGHJ6*02, IGHJ6*03 and IGHJ6*04. In another embodiment, the human JHgene segment is selected from IGHJ6*01, IGHJ6*02 and IGHJ6*04. Inanother embodiment, the JH gene segment is IGHJ6*02.

In a further embodiment, the human VH gene segment is IGHV3-23, forexample selected from IGHV3-23*01, IGHV3-23*02, IGHV3-23*03, IGHV3-23*04or IGHV3-23*05. In another embodiment, the human VH gene segment isIGHV3-23*01 or IGHV3-23*04, in particular IGHV3-23*04.

In a further embodiment, the human DH gene segment is IGHD3-10, forexample selected from IGHD3-10*01 or IGHD3-10*02. In one embodiment, thehuman DH gene segment is IGHD3-10*01. In one embodiment, the human DHgene segment is IGHD3-10*02.

-   77. The antibody or fragment according to any one of aspects 73 to    76, the VH domain comprising the HCDR1 sequence of SEQ ID NO:36 or    42 or the HCDR1 sequence of SEQ ID NO:36 or 42 comprising less than    4 amino acid substitutions.

In an embodiment, the HCDR1 sequence of SEQ ID NO:36 or 42 comprisesless than 3 amino acid substitutions (i.e. 2 or 1 substitutions). In anembodiment, the HCDR1 sequence of SEQ ID NO:36 or 42 comprises less than2 amino acid substitutions (i.e. one substitution).

-   78. The antibody or fragment according to any one of aspects 73 to    77, the VH domain comprising the HCDR2 sequence of SEQ ID NO:38 or    44, or the HCDR2 sequence of SEQ ID NO:38 or 44 comprising less than    5 amino acid substitutions.

In an embodiment, the HCDR2 sequence of SEQ ID NO:38 or 44 comprisesless than 4 amino acid substitutions (i.e. 3 or fewer). In anembodiment, the HCDR2 sequence of SEQ ID NO:38 or 44 comprises less than3 amino acid substitutions (i.e. 2 or 1 substitutions). In anembodiment, the HCDR2 sequence of SEQ ID NO:38 or 44 comprises less than2 amino acid substitutions (i.e. one substitution).

-   79. The antibody or fragment according to any one of aspects 73 to    78, the VH domain comprising an amino acid sequence of SEQ ID NO:    34, or a heavy chain variable domain amino acid sequence that is at    least 80% (e.g. at least 85%) identical to SEQ ID NO:34.

In an embodiment, the heavy chain variable domain amino acid sequence isat least 85%, at least 90%, at least 95%, least 96% at least 97% atleast 98% or at least 99% identical to SEQ ID NO:34.

-   80. The antibody or fragment according to any one of aspects 73 to    79 comprising first and second copies of said VH domain.-   81. The antibody or fragment according to any one of aspects 73 to    80, comprising a VL domain which comprises the LCDR1 sequence of SEQ    ID NO:54 or 60, or the LCRD3 sequence of SEQ ID NO:54 or 60    comprising less than 5 amino acid substitutions.

In an embodiment, the LCRD3 sequence of SEQ ID NO:54 or 60 comprisesless than 4 amino acid substitutions (i.e. 3 or fewer). In anembodiment, the LCRD3 sequence of SEQ ID NO:54 or 60 comprises less than3 amino acid substitutions (i.e. 2 or 1 substitutions). In anembodiment, the LCRD3 sequence of SEQ ID NO:54 or 60 comprises less than2 amino acid substitutions (i.e. one substitution).

-   82. The antibody or fragment according to any one of aspects 73 to    81, comprising a or said VL domain, which VL domain comprises the    LCDR2 sequence of SEQ ID NO:52 or 58, or the LCRD2 sequence of SEQ    ID NO:52 or 58 comprising less than 2 amino acid substitutions.-   83. The antibody or fragment according to any one of aspects 73 to    82, comprising a or said VL domain, which VL domain comprises the    LCDR1 sequence of SEQ ID NO:54 or 60, or the LCRD1 sequence of SEQ    ID NO:54 or 60 comprising less than 4 amino acid substitutions.

In an embodiment, the LCDR1 sequence of SEQ ID NO:54 or 60 comprisesless than 3 amino acid substitutions (i.e. 2 or 1 substitutions). In anembodiment, the LCDR1 sequence of SEQ ID NO:54 or 60 comprises less than2 amino acid substitutions (i.e. one substitution).

-   84. The antibody or fragment according to any one of aspects 73 to    83, comprising a or said VL domain, which VL domain comprises an    amino acid sequence of SEQ ID NOs: 48, or a light chain variable    domain amino acid sequence that is at least 80% (e.g. at least 85%)    identical to SEQ ID NO:48.

In an embodiment, the light chain variable domain amino acid sequence isat least 85%, at least 90%, at least 95%, least 96% at least 97% atleast 98% or at least 99% identical to SEQ ID NO:48.

-   85. The antibody or fragment according to any one of aspects 81 to    84, comprising first and second copies of said VL domain.-   86. The antibody or fragment according to any one of aspects 81 to    85, wherein the antibody or fragment comprises a kappa light chain.

In another embodiment, the VL domain is a kappa VL domain. In anembodiment, the kappa VL domain is derived from the recombination of ahuman VL gene segment, and a human JL gene segment, wherein the human VLgene segment is IGKV1D-39. In another embodiment, the VL gene segment isIGKV1D-39*01.

In a further embodiment, the human JL gene segment is IGKJ1 or IGKJ3. Inanother embodiment, the JL gene segment is IGKJ1*01. In anotherembodiment, the JL gene segment is IGKJ3*01.

-   87. The antibody or fragment according to any one of aspects 75 to    86 wherein the amino acid substitutions are conservative amino acid    substitutions, optionally wherein the conservative substitutions are    from one of six groups (each group containing amino acids that are    conservative substitutions for one another) selected from:    -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

In an embodiment, the conservative amino acid substitutions are asdescribed herein. For example, the substitution may be of Y with F, Twith S or K, P with A, E with D or Q, N with D or G, R with K, G with Nor A, T with S or K, D with N or E, I with L or V, F with Y, S with T orA, R with K, G with N or A, K with R, A with S, K or P. In anotherembodiment, the conservative amino acid substitutions may be wherein Yis substituted with F, T with A or S, I with L or V, W with Y, M with L,N with D, G with A, T with A or S, D with N, I with L or V, F with Y orL, S with A or T and A with S, G, T or V.

-   88. The antibody or fragment according to any one of aspects 73 to    87, wherein the antibody or fragment comprises a constant region,    e.g. an IgG4 constant region, optionally wherein the constant region    is IgG4-PE (Seq ID No:128).

In another example of any aspect herein, the antibody of fragmentcomprises a human gamma 4 constant region. In another embodiment, theheavy chain constant region does not bind Fc-γ receptors, and e.g.comprises a Leu235Glu mutation (i.e. where the wild type leucine residueis mutated to a glutamic acid residue). In another embodiment, the heavychain constant region comprises a Ser228Pro mutation to increasestability.

-   89. The antibody according to any one of aspects 73 to 88, wherein    the antibody comprises a heavy chain and a light chain, the heavy    chain amino acid sequence consisting of the sequence of SEQ ID No:62    and the light chain amino acid sequence consisting of the sequence    of SEQ ID No:64.-   90. An antibody or fragment as defined in any one of aspects 73 to    89, 98, 99, 101 or 102 for use in treating or preventing a    hOX40L-mediated disease or condition selected from an autoimmunc    disease or condition, a systemic inflammatory disease or condition,    or transplant rejection; for example inflammatory bowel disease    (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection,    allogeneic transplant rejection, graft-versus-host disease (GvHD),    ulcerative colitis, systemic lupus erythematosus (SLE), diabetes,    uveitis, ankylosing spondylitis, contact hypersensitivity, multiple    sclerosis or atherosclerosis, in particular GvHD.

The features of the antibodies, and the hOX40L-mediated disease of anyof the aspects, configurations, concepts, examples or embodiments asdescribed herein optionally apply mutatis mutandis to this use. Any ofthe compositions, dosing schedules or modes of administration asdescribed in any aspect, configuration, concept, example or embodimentherein optionally apply mutatis mutandis to this use.

-   91. Use of an antibody or fragment as defined in any one of aspects    73 to 89, 98, 99, 101 or 102 in the manufacture of a medicament for    administration to a human for treating or preventing a hOX40L    mediated disease or condition in the human selected from an    autoimmune disease or condition, a systemic inflammatory disease or    condition, or transplant/host rejection; for example inflammatory    bowel disease (IBD), Crohn's disease, rheumatoid arthritis,    transplant rejection, allogeneic transplant rejection,    graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus    erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis,    contact hypersensitivity, multiple sclerosis or atherosclerosis, in    particular GvHD.

The features of the antibodies, and the hOX40L-mediated disease of anyof the aspects, configurations, concepts, examples or embodiments asdescribed herein optionally apply mutatis mutandis to this use. Any ofthe compositions, dosing schedules or modes of administration asdescribed in any aspect, configuration, concept, example or embodimentherein optionally apply mutatis mutandis to this use.

-   92. A method of treating or preventing a hOX40L mediated disease or    condition selected from an autoimmune disease or condition, a    systemic inflammatory disease or condition, or transplant rejection;    for example inflammatory bowel disease (IBD), Crohn's disease,    rheumatoid arthritis, transplant rejection, allogeneic transplant    rejection, graft-versus-host disease (GvHD), ulcerative colitis,    systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing    spondylitis, contact hypersensitivity, multiple sclerosis or    atherosclerosis, in particular GvHD in a human, comprising    administering to said human a therapeutically effective amount of an    antibody or fragment as defined in any one of aspects 73 to 89, 98,    99, 101 or 102, wherein the hOX40L mediated disease or condition is    thereby treated or prevented.

The features of the antibodies, and the hOX40L-mediated disease of anyof the aspects, configurations, concepts, examples or embodiments asdescribed herein optionally apply mutatis mutandis to this method. Anyof the compositions, dosing schedules or modes of administration asdescribed in any aspect, configuration, concept, example or embodimentherein optionally apply mutatis mutandis to this method.

-   93. The antibody or fragment according to aspect 90, the use    according to aspect 91, or the method according to aspect 92,    wherein the hOX40L-mediated disease or condition is GvHD.

In another embodiment, the antibody or fragment is capable of treatingor preventing GvHD.

-   94. The antibody or fragment, the use or the method according to any    one of aspects 90 to 93, wherein the antibody is administered    prophylactically.

In an embodiment, the prophylaxis prevents the onset of the disease orcondition or of the symptoms of the disease or condition. In oneembodiment, the prophylactic treatment prevents the worsening, or onset,of the disease or condition. In one embodiment, the prophylactictreatment prevents the worsening of the disease or condition.

In another embodiment, said antibody is administered intravenously. Inanother embodiment, said antibody is administered at a dose of about5-10 mg/kg (e.g. at about 8 mg/kg). In another embodiment, said antibodyis administered at a dose selected from about 0.1 mg/kg, about 0.5mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, about 15 mg/kg,about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg about 90 mg/kg orabout 100 mg/kg, in particular about 1 mg/kg, or about 3 mg/kg.

In another embodiment, said antibody is administered 1-4 days beforetransplant, e.g. 1-3 days before transplant or 1-2 days beforetransplant. In another embodiment, said antibody is administered weekly,bi-weekly or monthly following transplant, e.g. bi-weekly. In a furtherembodiment, said antibody is administered intravenously prophylactically1-3 days before transplant at a dose of about 5-10 mg/kg (e.g. about 8mg/kg) and then intravenously, bi-weekly at a dose of about 5-10 mg/kg(e.g. about 8 mg/kg).

In another embodiment, the patient is monitored periodicallypost-transplant, for the presence of a biomarker predictive for thedevelopment of GvHD (e.g. acute GvHD), and the anti-OX40L antibody ofthe invention is administered once the biomarker levels are such thatthe patient is determined to be at risk of developing GvHD (e.g. acuteGvHD). This strategy would avoid unnecessary dosing of drug andunnecessary suppression of the immune system. Examples of biomarkerswhich may be useful as predictive biomarkers of acute GvHD may be thoseidentified in Levine et al., “A prognostic score for acutegraft-versus-host disease based on biomarkers: a multicentre study”,Lancet Haematol 2015; 2:e21-29. These biomarkers include, but are notlimited to TNFR1, ST-2, elafin and TL2Rα and Reg3α.

-   95. A human antibody or fragment thereof comprising a HCDR3 of from    16 to 27 amino acids and derived from the recombination of a human    VH gene segment, a human D gene segment and a human JH gene segment,    wherein the human JH gene segment is IGHJ6 (e.g. IGHJ6*02), which    specifically binds to hOX40L for treating or preventing a    hOX40L-mediated disease or condition selected from an autoimmune    disease or condition, a systemic inflammatory disease or condition,    or transplant rejection; for example inflammatory bowel disease    (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection,    allogeneic transplant rejection, graft-versus-host disease (GvHD),    ulcerative colitis, systemic lupus erythematosus (SLE), diabetes,    uveitis, ankylosing spondylitis, contact hypersensitivity, multiple    sclerosis or atherosclerosis, in particular GvHD (e.g. wherein the    antibody is for the prevention of GvHD).

The features of the antibodies, and the hOX40L-mediated disease of anyof the aspects, configurations, concepts, examples or embodimentsoptionally apply mutatis mutandis to this use. Any of the compositions,dosing schedules or modes of administration as described in any aspect,configuration, concept, example or embodiment herein optionally applymutatis mutandis to this use.

-   96. Use of a human antibody or fragment thereof comprising a HCDR3    of from 16 to 27 amino acids and derived from the recombination of a    human VH gene segment, a human D gene segment and a human JH gene    segment, wherein the human JH gene segment is IGHJ6 (e.g. IGHJ6*02),    which specifically binds to hOX40L in the manufacture of a    medicament for administration to a human for treating or preventing    a hOX40L mediated disease or condition in the human selected from an    autoimmune disease or condition, a systemic inflammatory disease or    condition, or transplant rejection; for example inflammatory bowel    disease (IBD), Crohn's disease, rheumatoid arthritis, transplant    rejection, allogeneic transplant rejection, graft-versus-host    disease (GvHD), ulcerative colitis, systemic lupus erythematosus    (SLE), diabetes, uveitis, ankylosing spondylitis, contact    hypersensitivity, multiple sclerosis or atherosclerosis, in    particular GvHD.

The features of the antibodies, and the hOX40L-mediated disease of anyof the aspects, configurations, concepts, examples or embodimentsoptionally apply mutatis mutandis to this use. Any of the compositions,dosing schedules or modes of administration as described in any aspect,configuration, concept, example or embodiment herein optionally applymutatis mutandis to this use.

-   97. A method of treating or preventing a hOX40L mediated disease or    condition selected from an autoimmune disease or condition, a    systemic inflammatory disease or condition, or transplant rejection;    for example inflammatory bowel disease (IBD), Crohn's disease,    rheumatoid arthritis, transplant rejection, allogeneic transplant    rejection, graft-versus-host disease (GvHD), ulcerative colitis,    systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing    spondylitis, contact hypersensitivity, multiple sclerosis or    atherosclerosis, in particular GvHD in a human, comprising    administering to said human a therapeutically effective amount of a    human antibody or fragment thereof comprising a HCDR3 of from 16 to    27 amino acids and derived from the recombination of a human VH gene    segment, a human D gene segment and a human JH gene segment, wherein    the human JH gene segment is IGHJ6 (e.g. IGHJ6*02), which    specifically binds to hOX40L, wherein the hOX40L mediated disease or    condition is thereby treated or prevented.

The features of the antibodies, and the hOX40L-mediated disease of anyof the aspects, configurations, concepts, examples or embodimentsoptionally apply mutatis mutandis to this method. Any of thecompositions, dosing schedules or modes of administration as describedin any aspect, configuration, concept, example or embodiment hereinoptionally apply mutatis mutandis to this method.

In an embodiment of any one of aspects 95 to 97, the human JH genesegment is selected from IGHJ6*01, IGHJ6*02, IGHJ6*03 and IGHJ6*04. Inanother embodiment of any one of aspects 95 to 97, the human JH genesegment is selected from IGHJ6*01, IGHJ6*02 and IGHJ6*04. In anotherembodiment of any one of aspects 95 to 97, the JH gene segment isIGHJ6*02.

In a further embodiment of any one of aspects 95 to 97, the human VHgene segment is IGHV3-23, for example selected from IGHV3-23*01,IGHV3-23*02, IGHV3-23*03, IGHV3-23*04 or IGHV3-23*05. In anotherembodiment of any one of aspects 95 to 97 the human VH gene segment isIGHV3-23*01 or IGHV3-23*04, in particular IGHV3-23*04.

In a further embodiment of any one of aspects 95 to 97, the human DHgene segment is IGHD3-10, for example selected from IGHD3-10*01 orIGHD3-10*02. In one embodiment of any one of aspects 95 to 97, the humanDH gene segment is IGHD3-10*01. In one embodiment of any one of aspects95 to 97, the human DH gene segment is IGHD3-10*02.

In an embodiment of any one of aspects 90 to 97, the antibody is capableof treating or preventing GvHD. In another embodiment of any one ofaspects 90 to 97, the antibody or fragment is used for the treatment orprevention of a disease other than GvD, but the antibody or fragment iscapable of treating or preventing GvHD.

-   98. The antibody or fragment according to aspect 86, or the antibody    or fragment according to aspect 95, the use according to aspect 96,    or the method according to aspect 97, wherein the antibody or    fragment comprises a kappa light chain, e.g. wherein the VL domain    of the light chain is derived from the recombination of a human VL    gene segment, and a human JL gene segment, wherein the human VL gene    segment is IGKV1D-39 (e.g. IGKV1D-39*01), and optionally the human    JL gene segment is IGKJ1 (e.g. IGKJ1*01) or IGKJ3 (e.g. IGKJ3*01).

In another embodiment, the VL domain is a kappa VL domain. In anembodiment, the kappa VL domain is derived from the recombination of ahuman VL gene segment, and a human JL gene segment, wherein the human VLgene segment is IGKV1D-39. In another embodiment, the VL gene segment isIGKV1D-39*01.

In a further embodiment, the human JL gene segment is IGKJ1. In anotherembodiment, the JL gene segment is IGKJ1*01. In a further embodiment,the human JL gene segment is IGKJ3. In another embodiment, the JL genesegment is IGKJ3*01

-   99. The antibody or fragment according to any one of aspects 73 to    89, 98, 101 or 102, or the antibody or fragment use or method    according to any one of aspects 90 to 98, wherein the antibody or    fragment enables greater than 80% stem cell donor chimerism by day    12 in a Rhesus macaque model of haploidentical hematopoietic stem    cell transplantation, optionally wherein the antibody is for the    prevention of GvHD.

In another aspect, there is provided an antibody or fragment, use ormethod according to any one of aspects 95 to 98, wherein the antibody orfragment is for treating or preventing transplant rejection (e.g. GvHD)in a human by enabling greater than 80% stem cell donor chimerism by day12 in said human following donor human hematopoietic stem celltransplantation.

In another embodiment, there is provided an antibody or fragmentaccording to any one of aspects 73 to 89, 98, 101 or 102, wherein theantibody or fragment enables greater than 80% stem cell donor chimerismby day 12 in a Rhesus macaque model of haploidentical hematopoietic stemcell transplantation.

In one embodiment, the chimerism is T cell (CD3⁺/CD20⁻) chimerism. Inanother embodiment, the chimerism is peripheral blood chimerism. Inanother embodiment, the chimerism is peripheral blood or T cell(CD3⁺/CD20⁻) chimerism.

In one embodiment, the stem cell donor chimerism (e.g. the peripheralblood or T cell (CD3⁺/CD20⁻) chimerism) is determined using divergentdonor- and recipient-specific MHC-linked microsatellite markers, bycomparing peak heights of the donor- and recipient-specific amplicons.In another embodiment, stem cell donor chimerism is determined asdescribed in Kean, L S, et al., “Induction of chimerism in rhesusmacaques through stem cell transplant and costimulation blockade-basedimmunosuppression”, Am J Transplant. 2007 February; 7(2):320-35. Inanother embodiment, stem cell donor chimerism is determined as describedin Example 7.

In one embodiment, the Rhesus macaque model of haploidenticalhaematopoietic stem cell is performed by the transplant (HSCT) recipientanimals undergoing a conditioning procedure together with anti-OX40Lantibody administration, followed by infusion of a peripheral bloodproduct isolated from a half-sibling donor animal, following whichanimals continue to receive weekly doses of the anti-OX40L antibody ofthe invention, and blood samples are taken and analysed for chimerism.

In another embodiment, in the HSCT model, recipient animals receive aconditioning radiation dose of 1020 cGy in 4 dose fractions over 2 days(experimental Day −2 and Day −1) to ablate the host haematopoieticsystem before intravenous administration of an anti-OX40L antibody ofthe invention (Day −2, with subsequent intravenous doses on Days 5, 12,19, 26, 33, 40, 47) and transplant of white blood cell- and stemcell-enriched peripheral blood from an MHC half-matched (half-sibling)donor animal to reconstitute the recipient's immune system, togetherwith provision of continuous supportive care, blood sampling andmonitoring for signs of GVHD.

In one embodiment, the antibody or fragment, use or method is for theprevention of GvHD.

In an embodiment, the anti-hOX40L antibody of the invention isadministered prophylactically. In one embodiment, the prophylactictreatment prevents the worsening or onset of the disease or condition.

In another embodiment, said antibody is administered intravenously. Inanother embodiment, said antibody is administered at a dose of about5-10 mg/kg (e.g. at about 8 mg/kg). In another embodiment, said antibodyis administered intravenously. In another embodiment, said antibody isadministered at a dose of about 5-10 mg/kg (e.g. at about 8 mg/kg). Inanother embodiment, said antibody is administered at a dose selectedfrom about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg,about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg,about 80 mg/kg about 90 mg/kg or about 100 mg/kg, in particular about 1mg/kg, or about 3 mg/kg.

In another embodiment, said antibody is administered 1-4 days beforetransplant, e.g. 1-3 days before transplant or 1-2 days beforetransplant. In another embodiment, said antibody is administered weekly,bi-weekly or monthly following transplant, e.g. bi-weekly. In a furtherembodiment, said antibody is administered intravenously prophylactically1-3 days before transplant at a dose of about 5-10 mg/kg (e.g. about 8mg/kg) and then intravenously, bi-weekly at a dose of about 5-10 mg/kg(e.g. about 8 mg/kg).

In another embodiment, the patient is monitored periodicallypost-transplant, for the presence of a biomarker predictive for thedevelopment of GvHD (e.g. acute GvHD), and the anti-OX40L antibody ofthe invention is administered once the biomarker levels are such thatthe patient is determined to be at risk of developing GvHD (e.g. acuteGvHD). This strategy would avoid unnecessary dosing of drug andunnecessary suppression of the immune system. Examples of biomarkerswhich may be useful as predictive biomarkers of acute GvHD may be thoseidentified in Levine et al., “A prognostic score for acutegraft-versus-host disease based on biomarkers: a multicentre study”,Lancet Haematol 2015; 2:e21-29. These biomarkers include, but are notlimited to TNFR1, ST-2, elafin and IL2Rα and Reg3α.

In a further embodiment, the HSCT model is conducted as described inMiller, Weston P., et al. “GVHD after haploidentical transplantation: anovel, MHC-defined rhesus macaque model identifies CD28⁻ CD8⁺ T cells asa reservoir of breakthrough T-cell proliferation during costimulationblockade and sirolimus-based immunosuppression.” Blood, 116,24(2010):5403-5418. In a further embodiment, the HSCT model is carriedout as described in Example 7.

-   100. The antibody or fragment, use or method according to any one of    aspects 95 to 99, wherein the antibody is as defined in any one of    aspects 73 to 89, 98, 99, 101 or 102.-   101. The antibody or fragment according to any one of aspects 73 to    89, 98, 99 or 102, or the antibody or fragment, use or method    according to any one of aspects 90 to 100, wherein the antibody or    fragment expresses as a stably transfected pool in Lonza GS-Xceed™    at level greater than 1.5 g/L in a fed batch overgrow culture using    Lonza version 8 feed system with an overgrow period of 14 days.

In one embodiment, the expression level is greater than 1.0 g/L, greaterthan 1.1 g/L, greater than 1.2 g/L, greater than 1.3 g/L or greater than1.4 g/L.

-   102. An antibody or fragment according to any one of aspects 73 to    89, 98, 99 or 101, or the antibody or fragment, use or method    according to any one of aspects 90 to 101, wherein the antibody or    fragment maintains a naïve population of CD4⁺ T-cells of >20% of    total CD4⁺ T cell population at day 12 in a Rhesus macaque model of    haploidentical hematopoietic stem cell transplantation.

In another aspect, there is provided an antibody or fragment accordingto any one of aspects 73 to 89, 98, 99 or 101, or an antibody orfragment, use or method according to any one of aspects 90 to 101,wherein the antibody or fragment is for treating or preventingtransplant rejection in a human by maintaining a naïve population ofdonor CD4⁺ T-cells of >20% of total CD4⁺ T cell population at day 12 insaid human following donor human hematopoietic stem cell transplantation

In one embodiment, the HSCT model is as described in any embodimentcontemplated hereinabove, e.g. as described in connection with aspect99.

In another embodiment, the naïve population is measured by evaluatingthe relative proportion of specific T cell phenotypes using flowcytometry where cell subsets are identified by labelling withfluorescent antibody probes and whereby naïve CD4 or CD8 T-cells arelabelled CD4⁺/CD28⁺/CD95⁻ or CD8⁺/CD28⁺/CD95⁻, respectively, centralmemory CD4 or CD8 T-cells are labelled CD4⁺/CD28⁺/CD95⁺ orCD8⁺/CD28⁺/CD95⁺, respectively, and effector memory CD4 or CD8 T-cellsare labelled CD4⁺/CD28⁻/CD95⁺ or CD8⁺/CD28⁻/CD95⁺, respectively.

-   103. The antibody or fragment, use or the method according to any    one of aspects 90 to 102, further comprising administering to the    human a further therapeutic agent, optionally wherein the further    therapeutic agent is independently selected from the group    consisting of rapamycin (sirolimus), tacrolimus, ciclosporin,    corticosteroids (e.g. methylprednisolone), methotrexate,    mycophenolate mofetil, anti-CD28 antibodies, anti-IL12/IL-23    antibodies (e.g. ustekinumab), anti-CD20 antibodies (e.g.    rituximab), anti-CD30 antibodies (e.g. brentuximab), CTLA4-Fc    molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.    maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.    natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52    antibodies (e.g. alemtuzumab), anti-CD45 antibodies,    cyclophosphamide, anti-thymocyte globulins, anti-complement C5    antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.    vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2R    antibodies (e.g. basilixumab), anti-CD25 antibodies (e.g.    daclizumab), anti-TNFa/TNFa-Fc molecules (e.g. etanercept,    adalimumab, infliximab, golimumab or certolizumab pegol) and    Vorinostat, in particular rapamycin (sirolimus), tacrolimus,    ciclosporin, corticosteroids (e.g. methylprednisolone),    methotrexate, mycophenolate mofetil, anti-CD28 antibodies, CTLA4-Fc    molecules (e.g. abatacept), anti-CD40L antibodies, anti-LFA1    antibodies, anti-CD52 antibodies (e.g. alemtuzumab),    cyclophosphamide and anti-thymocyte globulins.

In one embodiment, the further therapeutic agent is an anti-inflammatorydrug. In another embodiment, the anti-inflammatory drug is independentlyselected from the group consisting of corticosteroids (e.g.methylprednisolone), anti-IL12/IL-23 antibodies (e.g. ustekinumab),anti-VLA4 antibodies (e.g. natalizumab), anti-LFA1 antibodies,anti-complement C5 antibodies (e.g. eculizumab), anti-a4b7 integrinantibodies (e.g. vedolizumab), anti-IL6 antibodies (e.g. tocilizumab),anti-IL2R antibodies (e.g. basilixumab) or anti-TNFa antibodies/TNFa-Fcmolecules (e.g. etanercept, adalimumab, infliximab, golimumab,certolizumab pegol). In an example, the anti-inflammatory drug isindependently selected from the group consisting of corticosteroids(e.g. methylprednisolone) and anti-LFA1 antibodies.

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and further therapeutic agents independently selected fromthe group consisting of calcineurin inhibitors (e.g. tacrolimus,ciclosporin), mTOR inhibitors (e.g. rapamycin (sirolimus)), andantiproliferative agents (e.g. mycophenolate mofetil, cyclophosphamide).

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and further therapeutic agents independently selected fromthe group consisting of immunosuppressants that modulate IL-2 signalling(e.g. tacrolimus, ciclosporin, rapamycin (sirolimus), and anti-CD25antibodies (e.g. basilixumab, daclizumab).

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and rapamycin (sirolimus). In another embodiment, thecombination comprises an anti-OX40L antibody of the invention andtacrolimus. In another embodiment, the combination comprises ananti-OX40L antibody of the invention and tacrolimus and methotrexate. Inanother embodiment, the combination comprises an anti-OX40L antibody ofthe invention and ciclosporin. In another embodiment, the combinationcomprises an anti-OX40L antibody of the invention and ciclosporin andmethotrexate. In another embodiment, the combination comprises ananti-OX40L antibody of the invention and cyclophosphamide. In anotherembodiment, the combination comprises an anti-OX40L antibody of theinvention and mycophenolate mofetil.

-   104. The antibody or fragment, use or the method according to aspect    103, wherein the further therapeutic agent is administered    sequentially or simultaneously with the anti-hOX40L antibody or    fragment.-   105. A pharmaceutical composition comprising an antibody of fragment    as defined in any one of aspects 73 to 89, 98, 99, 101 or 102 and a    pharmaceutically acceptable excipient, diluent or carrier and    optionally further comprising a further therapeutic agent    independently selected from the group consisting of rapamycin    (sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.    methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28    antibodies, anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-CD20    antibodies (e.g.rituximab), anti-CD30 antibodies (e.g. brentuximab),    CTLA4-Fc molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.    maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.    natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52    antibodies (e.g. alemtuzumab), anti-CD45 antibodies,    cyclophosphamide, anti-thymocyte globulins, anti-complement C5    antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.    vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2R    antibodies (e.g. basilixumab), anti-CD25 antibodies (e.g.    daclizumab), anti-TNFa/TNFa-Fc molecules (e.g. etanercept,    adalimumab, infliximab, golimumab or certolizumab pegol) and    Vorinostat, in particular rapamycin (sirolimus), tacrolimus,    ciclosporin, corticosteroids (e.g. methylprednisolone),    methotrexate, mycophenolate mofetil, anti-CD28 antibodies, CTLA4-Fc    molecules (e.g. abatacept), anti-CD40L antibodies, anti-LFA1    antibodies, anti-CD52 antibodies (e.g. alemtuzumab),    cyclophosphamide and anti-thymocyte globulins.

The pharmaceutically acceptable excipients, diluents or carriers asdescribed herein apply mutatis mutandis to these compositions.

In one embodiment, the further therapeutic agent is an anti-inflammatorydrug. In another embodiment, the anti-inflammatory drug is independentlyselected from the group consisting of corticosteroids (e.g.methylprednisolone), anti-IL12/IL-23 antibodies (e.g. ustekinumab),anti-VLA4 antibodies (e.g. natalizumab), anti-LFA1 antibodies,anti-complement C5 antibodies (e.g. eculizumab), anti-a4b7 integrinantibodies (e.g. vedolizumab), anti-IL6 antibodies (e.g. tocilizumab),anti-IL2R antibodies (e.g. basilixumab) or anti-TNFa antibodies/TNFa-Fcmolecules (e.g. etanercept, adalimumab, infliximab, golimumab,certolizumab pegol). In an example, the anti-inflammatory drug isindependently selected from the group consisting of corticosteroids(e.g. methylprednisolone) and anti-LFA1 antibodies.

In one embodiment, the further therapeutic agent is independentlyselected from the group consisting of calcineurin inhibitors (e.g.tacrolimus, ciclosporin), mTOR inhibitors (e.g. rapamycin (sirolimus)),and antiproliferative agents (e.g. mycophenolate mofetil,cyclophosphamide).

In one embodiment, the further therapeutic agent is independentlyselected from the group consisting of immunosuppressants that modulateIL-2 signalling (e.g. tacrolimus, ciclosporin, rapamycin (sirolimus),and anti-CD25 antibodies (e.g. basilixumab, daclizumab).

In one embodiment, the further therapeutic agent is rapamycin(sirolimus). In another embodiment, the further therapeutic agent istacrolimus. In another embodiment, the further therapeutic agent is acombination of tacrolimus and methotrexate. In another embodiment, thefurther therapeutic agent is ciclosporin. In another embodiment, thefurther therapeutic agent is a combination of ciclosporin andmethotrexate. In another embodiment, the further therapeutic agent iscyclophosphamide. In another embodiment, the further therapeutic agentis mycophenolate mofetil

-   106. A pharmaceutical composition according to aspect 105, or a kit    comprising a pharmaceutical composition as defined in aspect 105,    wherein the composition is for treating and/or preventing a    hOX40L-mediated condition or disease selected from an autoimmune    disease or condition, a systemic inflammatory disease or condition,    or transplant rejection; for example inflammatory bowel disease    (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection,    allogeneic transplant rejection, graft-versus-host disease (GvHD),    ulcerative colitis, systemic lupus erythematosus (SLE), diabetes,    uveitis, ankylosing spondylitis, contact hypersensitivity, multiple    sclerosis and atherosclerosis, in particular GvHD.

The hOX40L-mediated diseases of any of the aspects, configurations,concepts, examples or embodiments described herein optionally applymutatis mutandis to this combination.

-   107. A pharmaceutical composition according to aspect 105 or aspect    106 in combination with, or kit according to aspect 106 comprising a    label or instructions for use to treat and/or prevent said disease    or condition in a human; optionally wherein the label or    instructions comprise a marketing authorisation number (e.g., an FDA    or EMA authorisation number); optionally wherein the kit comprises    an IV or injection device that comprises the antibody or fragment.

The labels, instructions, hOX40L-mediated diseases and conditions of anyof the aspects, configurations, concepts, examples or embodimentsdescribed herein optionally apply mutatis mutandis to this combination.

-   108. A nucleic acid that encodes the HCDR3 of an antibody or    fragment as defined in any one of aspects 73 to 89, 98, 99, 101 or    102.-   109. A nucleic acid that encodes a VH domain and/or a VL domain of    an antibody or fragment as defined in any one of aspects 73 to 89,    98, 99, 101 or 102.-   110. A nucleic acid according to aspect 109 comprising a nucleotide    sequence that is at least 80% identical to the sequence of SEQ ID    NO: 33 and/or SEQ ID NO: 47.

In an example, the nucleotide sequence is at least 85% identical, atleast 90% identical, at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical or at least 99% identical tothe sequence of SEQ ID NO: 33 and/or SEQ ID NO: 47.

-   111. A nucleic acid that encodes a heavy chain or a light chain of    an antibody recited in any one of aspects 73 to 89, 98, 99, 101 or    102.-   112. A vector comprising the nucleic acid of any one of aspects 108    to 111; optionally wherein the vector is a CHO or HEK293 vector.-   113. A host comprising the nucleic acid of any one of aspects 108 to    111 or the vector of aspect 112.

The present invention furthermore relates to the following concepts:

Concept 1. A method of reducing the proportion of (e.g. of depleting ordecreasing the level of) CD45RA+CCR7+CD95+OX40+ memory stem T-cells(Tscm) comprising combining said cells with an agent (such as ananti-OX40 or an anti-OX40L antibody or fragment thereof) which reducesthe proportion of Tscm cells (e.g. which depletes or decreases the levelof said Tscm cells), and whereby the proportion of said Tscm cells isreduced (e.g. whereby the level of said Tscm cells is decreased ordepleted).

CD45RA+CCR7+CD95+OX40+ memory stem T-cells (Tscm) are thought to be anewly-defined subset of stem cell memory T-cells which are long-livedand have the capacity for self-renewal. Therefore, these cells may bedetrimental in various diseases, such as GvHD and autoimmune disorders,because they are the source of a persistent and multipotent populationof potentially self-reactive effector T-cells. It is known that T-cellsdevelop through a pathway beginning with naïve T-cells (Tn), throughstem cell memory T-cells (Tscm), through central memory T-cells (Tcm)and effector memory T-cells (Tem), before developing into short-livedeffector T-cells (Teff), as described in Gattinoni and Restifo (2013),Inside Blood, 121(4), 567-568. At these various stages, differentmarkers are expressed on the surface of the T-cells, which reflect theactivation status of the T-cells, their tissue localisation and theirresponsiveness to various stimuli such as inflammatory cytokines.

Tscm cells as defined herein are characterised asCD45RA+CCR7+CD95+OX40+. For alternative prior art classifications ofdifferent T-cell types, see the figure in Gattinoni and Restifo (2013).Further markers may be present or absent, but Tscm cells must at aminimum be CD45RA+CCR7+CD95+OX40+. The various cell-surface markers are,in one embodiment, identified using flow cytometry using methods whichare well-known to those of skill in the art. In one embodiment, the Tscmcells may additionally be CD8+. In another embodiment, the Tscm cellsmay additionally be CD62L+. Flow cytometry techniques are well-known tothose skilled in the art. Agents which may be used in flow cytometrytechniques are defined in Example 7 below. In one embodiment, the flowcytometry is carried out as described in Example 7 below. In anotherembodiment, the flow cytometry is carried out as described in Baumgarth& Roederer (2000), Journal of Immunological Methods, 243, 77-97. (seeconcept 25 hereinbelow).

In a particular embodiment, the Tscm cells are characterised as beingCD4+CD45RA+CCR7+CD95+OX40+.

Without being bound by theory, it is thought that reducing this Tscmpopulation will have a number of benefits in various diseases, as setout herein. In one embodiment, the Tscm cells are active Tscm cells.

Throughout concepts 1 to 83 herein, the proportion or levels of Tscmcells may be reduced in a sample, or indeed in (a sample of) the bloodof a subject. The proportion or levels of Tscm cells may be determinedrelative to the entire T-cell population in the sample. In oneembodiment, the proportion or level of Tscm cells is determined relativeto other T-cells in the sample. T-cells generally may be identified asbeing CD3+, and include Tn cells, Tscm cells, Tcm cells, Tem cells andTeff cells. In a particular embodiment, the proportion or level of Tscmcells is determined relative to Tn cells (as defined hereinbelow) in thesample. The proportion or level of Tscm cells may be altered bydepletion or by a decrease. In one embodiment, a ratio of T-cell typesmay be the same as a proportion of Tscm cells (e.g. as for concept 2hereinbelow). In another embodiment, a level of T-cell types may be thesame as a proportion of Tscm cells. In one embodiment, the ratio orproportion of Tscm:Tn is greater than 50:50. Particular ratios andproportions are as described in concepts 23 and 24.

As used in concepts 1 to 83 herein “depleting” and “depletes” describesan active effect following combination with an agent (such as anantibody) on the desired target to kill or remove the target cells (e.g.Tscm cells). When the agent is an antibody, this is usually achievedthrough effector functions, such as ADC, ADCC or CDC. Alternatively, thetarget may be killed or removed by a toxin, which may be conjugated to adrug or targeting moiety (such as an anti-OX40 or an anti-OX40Lantibody). Such toxins will selectively kill or remove the cell to whichthey are targeted. Suitable immunoconjugates are described on page 90,and on pages 114 to 118, and 134 (in particular pages 114 to 116)herein.

“Decreasing” or “decreases” as used in concepts 1 to 83 herein refers toa mechanism other than depletion, which reduces the absolute number ofcells in a given population. This may be achieved indirectly, forexample through a blocking or neutralising agent (such as an antibody)against a target which indirectly results in the killing of a targetcell (such as a Tscm), or prevents the expansion or growth of the targetcells, resulting in an apparent decrease in proportions relative toanother type of cell (such as Tn cells).

As used in concepts 1 to 83 herein, a “level” of a T-cell population mayrefer to the absolute number, or to the relative proportion of a type ofT-cell.

Throughout the various concepts 1 to 83 described herein, an agent whichreduces the proportion of Tscm cells may be, for example, an antibody orfragment thereof, a short interfering RNA (SiRNA), a zinc finger, aDARPin, an aptamer, a Spiegelmer, an ant-calin, a receptor-Fc fusion, aligand-Fc fusion or a small molecule. In one embodiment, the agenttargets OX40 (e.g. human OX40), or ligands of OX40. In anotherembodiment, the agent targets OX40L (e.g. human OX40L), or receptors ofOX40L. In one example, the agent may be an OX40-Fc fusion protein (e.g.hOX40-Fc fusion), or may be an OX40L-Fc fusion protein (e.g. hOX40L-Fcfusion), both including functional fragments of OX40 and OX40L. Thesetypes of constructs are known to those skilled in the art. In anotherembodiment, the agent targets OX40 (e.g. human OX40). In anotherembodiment, the agent targets OX40L (e.g. human OX40L).

In a particular embodiment, the agent is an antibody or fragmentthereof. Formats and structures of antibodies and fragments aredescribed elsewhere herein and may be applied to any of the conceptsdisclosed herein. The antibody or fragment may be any of the constructsas described herein (for example, as in any one of concepts 52 to 64herein). In a particular embodiment, the agent is an anti-human OX40antibody or fragment thereof. In another particular embodiment, theagent is an anti-human OX40L antibody, such as an antibody comprisingthe amino acid sequence of 02D10 described herein or an antibodycomprising the amino acid sequence of oxelumab.

Concept 2. A method of altering the ratio of cell types in a T-cellpopulation in a sample, the method comprising:

-   -   a. providing said population, wherein the population comprises a        mixture of different T-cell types, wherein the population        comprises CD45RA+CCR7+CD95+OX40+ Tscm cells,    -   b. providing an agent which reduces the proportion of Tscm cells        (or providing an anti-OX40 or an anti-OX40L antibody or fragment        thereof); and    -   c. combining said cell population with an amount of said agent        (e.g. antibody or fragment thereof) effective to alter the ratio        (e.g. to reduce the proportion) of Tscm cells in said        population.

Throughout concepts 1 to 83 herein, the ratio of T-cell types may bealtered in a sample, for example by increasing the proportion of naïveT-cells (Tn, as defined hereinbelow). In another embodiment, the ratioof T-cell types may be altered by decreasing the proportion of Tscmcells. The ratio of Tscm cells may be determined relative to the entiresample. In one embodiment, the ratio of T-cells is determined bycomparing the proportion of naïve T-cells or Tscm cells relative toother T-cells in the sample. T-cells generally may be identified asbeing CD3+, and include Tn cells, Tscm cells, Tcm cells, Tem cells andTeff cells. In a particular embodiment, the ratio of T-cells isdetermined as the ratio of Tscm cells relative to naïve T-cells in thesample. The ratio of T-cells may be altered by depletion or by adecrease of Tscm cells. The ratio of T-cells may be altered by anincrease or expansion of naïve T-cells.

Concept 3. A method according to concept 2, wherein in step a), thepopulation further comprises CD45RA+CCR7+CD95− naïve T-cells (Tn).

Naïve T-cells (Tn) as defined in concepts 1 to 83 herein arecharacterised as CD45RA+CCR7+CD95−. Further markers may be present orabsent, but Tn cells must at a minimum be CD45RA+CCR7+CD95−. In oneembodiment, Tn cells may additionally be CD8+ or CD4+, in particularCD4+. It is thought that Tn are beneficial because these represent theentire pool of T-cells from which adaptive T-cell immune responses candevelop to protect an individual when exposed to potentially harmfulpathogens and malignant cells.

Concept 4. A method according to concept 3 wherein the ratio of Tscm:Tnin the population of step a) is greater than 50:50.

Concept 5. A method according to any one of concepts 1 to 4, wherein themethod is carried out ex vivo in a sample of blood extracted from ahuman donor subject.

Concept 6. A method according to concept 5, wherein blood produced bysaid method is reintroduced to a recipient human subject.

In one embodiment, the recipient human subject is the same donor humansubject from whom the sample was removed. In another embodiment, therecipient human subject is different to the donor human subject. Whenthe recipient is different to the donor, it is preferable that the donoris of the same gender as the recipient subject. In another embodiment,the donor may be of a similar age and ethnicity as the recipientsubject. In another embodiment, the donor may have the same or similarallotype markers as the recipient subject.

In another embodiment, the recipient human donor may receive more thanone transfusion of donor blood, according to the severity of the diseaseto be treated.

Concept 7. A method according to any one of concepts 1 to 4, wherein themethod is carried out in vivo in a human subject.

Concept 8. A method according to concept 7, wherein the subject has oris at risk of a Tscm-mediated disease or condition.

As used herein, a subject may be identified as being “at risk of aTscm-mediated disease or condition” when the cellular changes in theirT-cell population have begun to take place, but the subject has not yetpresented symptoms or would not be diagnosed as having such a disease byany conventional method. Thus, the methods and uses disclosed herein mayaid in the early identification of patients who will develop suchdiseases. In one embodiment, the disease is prevented (i.e. thetreatment is prophylactic).

In a particular embodiment, the subject is at risk of GvHD or transplantrejection when they are pre-operative for a transplant. Potentialtransplant therapies are envisaged in concept 78 hereinbelow.

In any of concepts 1 to 83 described herein, a Tscm-mediated disease maybe as defined in any of concepts 71 to 80 hereinbelow.

Concept 9. A method of treating or reducing the risk of a Tscm-mediateddisease or condition in a subject, the method comprising combining apopulation of T-cells with an agent (e.g. an anti-OX40 or an anti-OX40Lantibody or fragment thereof) which reduces the proportion of Tscm cells(e.g. which depletes or decreases the level of said Tscm cells), andwhereby the proportion of CD45RA+CCR7+CD95+OX40+ Tscm cells is reducedin the population (e.g. whereby the level of said Tscm cells isdecreased or depleted in said population).

As used in concepts 1 to 83 herein, the “treatment” of a Tscm-mediateddisease includes the reduction of one or more symptom(s) of saidTscm-mediated disease. The “prevention” of a Tscm-mediated diseaseincludes the prevention of one or more symptom(s) of said Tscm-mediateddisease.

Concept 10. A method according to any one of concepts 7 to 9, whereinthe agent (e.g. antibody or fragment thereof) is combined byadministering said agent (e.g. antibody or fragment) in atherapeutically effective amount to said subject, whereby saidTscm-mediated disease or condition is treated or the risk of saidTscm-mediated disease or condition is reduced in said subject.

In one embodiment, the administration is prophylactic to reduce the riskof a Tscm-mediated disease.

In any of the concepts described herein, a therapeutically effective orprophylactically effective amount of the antibody or fragment is asdescribed elsewhere (see page 29, 73, 105 to 107 for therapy, and pages72, and 102 to 103 for prophylaxis). In any of the concepts describedherein, modes and compositions for administration may be as describedelsewhere (see pages 118 to 142 herein). In one embodiment, the antibodyor fragment is administered by bolus injection (e.g. intravenously).

Concept 11. A method of treating or reducing the risk of a Tscm-mediateddisease or condition in a subject comprising administering to saidsubject a therapeutically effective amount of an agent (e.g. ananti-OX40 or an anti-OX40L antibody or fragment thereof) which reducesthe proportion of Tscm cells (e.g. which depletes or decreases the levelof said Tscm cells), and whereby the proportion ofCD45RA+CCR7+CD95+OX40+ Tscm cells is reduced (e.g. whereby the level ofsaid Tscm cells is decreased or depleted), wherein the Tscm-mediateddisease or condition is thereby treated or the risk of saidTscm-mediated disease or condition is reduced.

Concept 12a. An agent (e.g. an anti-OX40 or an anti-OX40L antibody orfragment thereof) which reduces the proportion of Tscm cells (e.g. whichdepletes or decreases the level of said Tscm cells) for use in treatingor reducing the risk of a Tscm-mediated disease or condition in asubject; or concept 12b. An anti-OX40 or an anti-OX40L antibody orfragment thereof for use in treating or reducing the risk of aTscm-mediated disease or condition in a subject.

Concept 13a. Use of an agent (e.g. an anti-OX40 or an anti-OX40Lantibody or fragment thereof) which reduces the proportion of Tscm cells(e.g. which depletes or decreases the level of said Tscm cells) for thetreatment or prevention of a Tscm-mediated disease or condition in asubject; or concept 13b. Use of an anti-OX40 or an anti-OX40L antibodyor fragment thereof for the treatment or prevention of a Tscm-mediateddisease or condition in a subject.

Concept 14a. Use of an agent (e.g. an anti-OX40 or an anti-OX40Lantibody or fragment thereof) which reduces the proportion of Tscm cells(e.g. which depletes or decreases the level of said Tscm cells) in themanufacture of a medicament for the treatment or prevention of aTscm-mediated disease or condition in a subject; or concept 14b. The useof an anti-OX40 or an anti-OX40L antibody or fragment thereof in themanufacture of a medicament for the treatment or prevention of aTscm-mediated disease or condition in a subject.

Concept 15a. A composition comprising an agent (e.g. an anti-OX40 or ananti-OX40L antibody or fragment thereof) which reduces the proportion ofTscm cells (e.g. which depletes or decreases the level of said Tscmcells) for the treatment or prevention of a Tscm-mediated disease orcondition in a subject; or concept 15b. A composition comprising ananti-OX40 or an anti-OX40L antibody or fragment thereof for thetreatment or prevention of a Tscm-mediated disease or condition in asubject.

Concept 16. A method of treating a disease or condition in a subject inneed thereof, comprising:

-   -   a. Performing an assay to measure the level of CD45RA+CCR7+CD95−        Tn cells and the level of CD45RA+CCR7+CD95+OX40+ Tscm cells in a        sample obtained from the subject; and    -   b. Administering an agent (e.g. an anti-OX40 or an anti-OX40L        antibody or fragment thereof) which reduces the proportion of        Tscm cells (e.g. which depletes or decreases the level of said        Tscm cells), such as an anti-OX40 or an anti-OX40L antibody or        fragment thereof, to the subject when the ratio of Tscm:Tn cells        in the sample is determined in the assay to be greater than        50:50.

Concept 17a. An agent (e.g. an anti-OX40 or an anti-OX40L antibody orfragment thereof) which reduces the proportion of Tscm cells (e.g. whichdepletes or decreases the level of said Tscm cells) for use in therapyof a subject, wherein the agent is to be administered to a subject whohas, or has been determined to have, a ratio of CD45RA+CCR7+CD95+OX40+Tscm cells:CD45RA+CCR7+CD95− Tn cells of greater than 50:50; or concept17b. An anti-OX40 or an anti-OX40L antibody or fragment thereof for usein therapy of a subject, wherein the antibody or fragment thereof is tobe administered to a subject who has, or has been determined to have, aratio of CD45RA+CCR7+CD95+OX40+ Tscm cells:CD45RA+CCR7+CD95− Tn cells ofgreater than 50:50.

Concept 18a. Use of an agent (e.g. an anti-OX40 or an anti-OX40Lantibody or fragment thereof) which reduces the proportion of Tscm cells(e.g. which depletes or decreases the level of said Tscm cells) fortherapy of a subject who has, or has been determined to have, a ratio ofCD45RA+CCR7+CD95+OX40+ Tscm cells: CD45RA+CCR7+CD95− Tn cells of greaterthan 50:50; or concept 18b. Use of an anti-OX40 or an anti-OX40Lantibody or fragment thereof for therapy of a subject who has, or hasbeen determined to have, a ratio of CD45RA+CCR7+CD95+OX40+ Tscm cells:CD45RA+CCR7+CD95− Tn cells of greater than 50:50.

Concept 19a. Use of an agent (e.g. an anti-OX40 or an anti-OX40Lantibody or fragment thereof) which reduces the proportion of Tscm cells(e.g. which depletes or decreases the level of said Tscm cells) in themanufacture of a medicament for use in therapy of a subject, wherein theagent is to be administered to a subject who has, or has been determinedto have, a ratio of CD45RA+CCR7+CD95+OX40+ Tscm cells:CD45RA+CCR7+CD95−Tn cells of greater than 50:50; or concept 19b. Use of an anti-OX40 oran anti-OX40L antibody or fragment thereof in the manufacture of amedicament for use in therapy of a subject, wherein the antibody orfragment thereof is to be administered to a subject who has, or has beendetermined to have, a ratio of CD45RA+CCR7+CD95+OX40+ Tscmcells:CD45RA+CCR7+CD95− Tn cells of greater than 50:50.

In any of concepts 17 to 19, the ratio is determined in a sample, forexample, in a sample of blood obtained from said subject.

Concept 20. A method according to concept 16a orb, an agent or anantibody or fragment for the use according to concept 17a or b, or theuse according to concept 18a or b or concept 19 a or b, wherein thetherapy is the treatment or prevention of a Tscm-mediated disease orcondition, preferably wherein the therapy is the treatment of aTscm-mediated disease or condition.

In another embodiment, the subject has or is at risk of a Tscm-mediateddisease or condition. The Tscm-mediated disease or condition may be asdefined in any one of concepts 71 to 80 hereinbelow.

Concept 21. A method of classifying a subject as having or as being atrisk of a Tscm-mediated disease or condition (e.g. which disease orcondition is suitable for treatment with an anti-OX40 or an anti-OX40Lantibody or fragment thereof), comprising:

-   -   a. performing an assay that detects (i) CD45RA+CCR7+CD95+OX40+        Tscm cells, and (ii) CD45RA+CCR7+CD95− Tn cells in a sample        obtained from said subject; and    -   b. classifying the subject as having, or as being at risk of a        Tscm-mediated disease or condition if the ratio of Tscm:Tn cells        in the sample is greater than 50:50.

Concept 22. A method according to concept 21 further comprising the stepof:

-   -   c. administering to said subject an anti-OX40 or an anti-OX40L        antibody or fragment thereof which reduces the proportion of        said Tscm cells in the blood of said subject, (e.g. which        depletes or decreases the level of said Tscm cells) if said        subject has been classified as having or as being at risk of a        Tscm-mediated disease or condition in step b).

Tscm-mediated diseases or conditions which may be suitable for treatmentwith an anti-OX40 or an anti-OX40L antibody or fragment thereof are asdescribed in any one of concepts 71 to 80 herein below.

Concept 23. A method according to any one of concepts 4, 16, or 20 to22, an agent or an antibody or fragment for the use according to concept17 or 20, or the use according to any one of concepts 18 to 20, whereinthe ratio of Tscm:Tn cells is (or is determined or classified to be)greater than 60:40, or is greater than 70:30, or is greater than 75:25,such as greater than 70:30.

In another embodiment, the ratio is (or is determined or classified tobe) greater than 55:45. In another embodiment, the ratio is (or isdetermined or classified to be) greater than 65:35.

Concept 24. A method, agent or an antibody or fragment for the use, orthe use according to concept 23, wherein the ratio of Tscm:Tn cells is(or is determined or classified to be) greater than 80:20, or is greaterthan 85:15, for example greater than 90:10, e.g. greater than 95:5.

Concept 25. A method according to any one of concepts 4, 16, or 20 to24, an agent or an antibody or fragment for the use according to any oneof concepts 17, 20, 23 or 24, or the use according to any one ofconcepts 18 to 20, 23 or 24, wherein the ratio of Tscm:Tn cells isdetermined (or is determinable) by flow cytometry.

Flow cytometry techniques are well-known to those skilled in the art, asdiscussed above. Agents which may be used in flow cytometry techniquesare defined in Example 7 below. In one embodiment, the flow cytometry iscarried out as described in Example 7 below. In another embodiment, theflow cytometry is carried out as described in Baumgarth & Roederer(2000).

Concept 26. A method for treating or reducing the risk of aTscm-mediated disease or condition with an agent (e.g. an anti-OX40 oran anti-OX40L antibody or fragment thereof) which reduces the proportionof Tscm cells (e.g. which depletes or decreases the level of said Tscmcells), or with an anti-OX40 or an anti-OX40L antibody or fragmentthereof, comprising the steps of:

-   -   a. determining whether the subject is a candidate for treatment        by detecting the presence of OX40 on the surface of        CD45RA+CCR7+CD95+ Tscm cells obtained from a sample from the        subject; and    -   b. administering said agent, such as said antibody or fragment,        to the subject if the subject is identified as a candidate for        treatment.

Concept 27. A method according to concept 26, wherein the presence ofOX40 on the surface of the Tscm cells is determined using flowcytometry.

In one embodiment, the subject is a human and the OX40 is human OX40.

Concept 28. A method, comprising:

-   -   a. obtaining at least two T-cell samples derived from a subject        who has or is at risk of a Tscm-mediated disease or condition,        wherein said at least two samples comprise a first sample and a        second sample,    -   b. determining levels of CD45RA+CCR7+CD95+OX40+ Tscm cells in        said first and second samples;    -   c. treating said subject to reduce the proportion of        CD45RA+CCR7+CD95+OX40+ Tscm cells (e.g. to deplete or decrease        the level of Tscm cells) by administering an agent which reduces        the proportion of Tscm cells (e.g. which depletes or decreases        the level of said Tscm cells), or by administering an anti-OX40        or an anti-OX40L antibody or fragment thereof, if the levels of        Tscm cells in said second sample are elevated as compared to        said first sample, in order to treat or reduce the risk of said        Tscm-mediated disease or condition.

The levels of Tscm in said first and second samples in step b. may beeither the absolute number of Tscm cells, or may be the relativeproportions of Tscm (e.g. the ratio of Tscm:Tn, or Tacm:total T-cellcount). The levels of Tscm cells may be elevated in the second sample ifthey are statistically significantly higher than the levels in the firstsample.

Concept 29. A method according to concept 28, wherein said first sampleis collected:

-   -   i. before the onset of said disease or condition; or    -   ii. after the onset of said disease or condition; and

optionally wherein said second sample is collected no longer than onemonth, e.g. no longer than one week after the first sample.

As used in the concepts herein, a subject may be determined to be“before the onset of a Tscm-mediated disease or condition” if thesubject is presenting no symptoms which would conventionally beassociated with said disease or condition or if the subject would not bediagnosed as having such a disease or condition by any conventionalmethod. For example, the presence of signs and symptoms of acute GvHDmay be staged and graded according to a standardised scale such asdescribed in Przepiorka et al. (1995), 1994 Consensus Conference onAcute GvHD Grading Bone Marrow Transplant 1995; 15, 825-828. Similardisease grading scales are also in routine clinical use for otherrelevant diseases, such as rheumatoid arthritis and inflammatory boweldiseases.

Concept 30. A method according to concept 28 or concept 29, wherein theTscm-mediated disease or condition is a transplant, and wherein in stepc) the treatment is in order to reduce the risk of transplant rejection,optionally wherein the first sample is taken before the transplant, andthe second sample is taken after the transplant.

The first sample may be taken pre-operatively, e.g. after the subjecthas been identified as a candidate for treatment. The second sample istaken after the transplant and may be used by physicians as a method ofmonitoring the acceptance of the transplant. Thus, it may be that thephysician may take more than one sample after the transplant, e.g. adaily blood sample to monitor the subject for changes in the proportionof Tscm:Tn or the levels of Tscm in the sample. The samples may be takenevery other day, weekly, monthly or longer (including yearly) accordingto the likelihood of transplant rejection. For example, if thetransplant is autologous, then the likelihood of transplant rejectionmay be reduced as compared to an allogeneic transplant, and thereforethe time period between sample collections post-transplant may be longerthan with an allogeneic transplant, where the risk of rejection ishigher.

Concept 31. A method according to concept 30, wherein in step a), thefirst sample is collected no longer than a week, e.g. no longer than 6days, no longer than 5 days, no longer than 4 days, or no longer than 3days, such as no longer than 2 days before said transplant.

Concept 32. A method according to any one of concepts 28 to 31, whereinthe second sample is collected no longer than 6 days, no longer than 5days, no longer than 4 days, or no longer than 3 days, such as no longerthan 2 days after the first sample or after said transplant.

Concept 33. A method according to any one of concepts 28 to 32, whereinin step c), the levels of Tscm cells in said second sample are greaterthan double the levels as compared to said first sample, for example aregreater than three times the level, or preferably are greater than 4times the levels as compared to said first sample.

In one embodiment, in step c), the levels of Tscm cells in said secondsample are greater than 4 times (e.g. greater than 4.5 times) the levelsas compared to said first sample. In another embodiment, in step c), thelevels of Tscm cells in said second sample are greater than 5 times thelevels as compared to said first sample.

Concept 34. A method according to any one of concepts 30 to 33, whereinthe subject is given a prophylactic dose of an agent which reduces theproportion of Tscm cells (e.g. which depletes or decreases the level ofsaid Tscm cells), or is given a prophylactic dose of an anti-OX40 or ananti-OX40L antibody or fragment thereof, before said transplant, and thefirst sample is taken before administration of said agent, or antibodyor fragment thereof, and wherein the second sample is taken after thetransplant or after administration of the agent, or the antibody orfragment thereof (preferably, where in the second sample is taken afterthe transplant).

In one embodiment, the prophylactic dose is an effective prophylacticdose. By “effective”, it is meant that the dose is effective to reducethe proportion or level of Tscm as described herein, or effective toprevent or reduce the risk of a Tscm-mediated disease or condition.

The methods as described herein may be used to correct analready-aberrant level of Tscm cells, by administration of an agentwhich reduces the proportion of Tscm cells (e.g. which depletes ordecreases the level of said Tscm cells), or by administration of ananti-OX40 or an anti-OX40L antibody or fragment thereof, before atransplant, in order to reduce the risk of transplant rejection afterthe transplant. Therefore, multiple samples may be taken afteradministration of the agent (or of the anti-OX40 or an anti-OX40Lantibody or fragment thereof), but before the transplant. Comparison maybe made between the collected samples and a sample obtained from ahealthy donor.

Concept 35. A method according to any one of concepts 30 to 33, whereinthe subject is given a therapeutic dose of an agent which reduces theproportion of Tscm cells (e.g. which depletes or decreases the level ofsaid Tscm cells), or is given a therapeutic dose of an anti-OX40 or ananti-OX40L antibody or fragment thereof, after the transplant, andwherein the first sample is taken before said transplant, and the secondsample is taken after the transplant.

In one embodiment, the therapeutic dose is an effective therapeuticdose. By “effective”, it is meant that the dose is effective to reducethe proportion or level of Tscm as described herein, or effective totreat a Tscm-mediated disease or condition.

In one embodiment, the second sample is taken after the administrationof the agent, or antibody or fragment thereof. This would enable aphysician to check that the levels or proportion of Tscm cells remain“normal”, i.e. as compared to the first sample, or to a sample obtainedfrom a healthy donor.

Concept 36. A method according to any one of concepts 30 to 33, whereinthe subject is given a therapeutic dose of an agent which reduces theproportion of Tscm cells (e.g. which depletes or decreases the level ofsaid Tscm cells), or is given a therapeutic dose of an anti-OX40 or ananti-OX40L antibody or fragment thereof, after the transplant, andwherein the first sample is taken before said transplant, and the secondsample is taken after the administration of said agent, or of saidantibody or fragment thereof.

Concept 37. A method according to any one of concepts 34 to 36, furthercomprising the steps of:

-   -   d. obtaining a third sample derived from said subject;    -   e. determining the levels of CD45RA+CCR7+CD95+OX40+ Tscm cells        in said third sample;    -   f. treating said subject to reduce the proportion of        CD45RA+CCR7+CD95+OX40+ Tscm cells (e.g. to deplete or decrease        the level) of Tscm cells by administering an agent which reduces        the proportion of Tscm cells (e.g. which depletes or decreases        the level of said Tscm cells), or by administering an anti-OX40        or an anti-OX40L antibody or fragment thereof, if the levels of        Tscm cells in said third sample are elevated as compared to said        second or said first sample.

The levels are considered to be “elevated” as described hereinabove(e.g. as for concept 28 or 33).

Concept 38. A method according to concept 37, wherein steps d) to f) arerepeated as necessary until the levels of Tscm cells remain at atherapeutically-effective, or at a prophylactically-effective levels,e.g. at a substantially constant level in said subject.

As used in the concepts herein, a “substantially constant level” may bedescribed as within 30% variance between samples. In one embodiment, asubstantially constant level is within 20% variance between samples. Inanother embodiment, a substantially constant level is within 15%variance between samples, such as within 10% variance between samples,e.g. within 5% variance between samples.

Concept 39. A method according to any one of concepts 28 to 38, whereinthe second sample is taken no longer than one month after the firstsample, such as no longer than one week, no longer than 6 days, nolonger than 5 days, no longer than 4 days, or no longer than 3 days,e.g. no longer than 2 days after the first sample, and optionallywherein the third sample is taken no longer than one month after thesecond sample, such as no longer than one week, no longer than 6 days,no longer than 5 days, no longer than 4 days, or no longer than 3 days,e.g. no longer than 2 days after the second sample.

Timepoints for taking any of the samples described in these conceptswill depend on a number of factors, such as the likelihood of thesubject having or being at risk of a Tscm-mediated disease (e.g. GvHD ortransplant rejection), the level determined in the previous sample, thetype of transplant, etc. A person skilled in the art will be able todetermine appropriate time points as necessary or desired. Thetimepoints may be monthly, every other month, quarterly, half-yearly oryearly, if desired.

Concept 40. In vitro use of CD45RA+CCR7+CD95+OX40+ Tscm cells, as adiagnostic for a Tscm-mediated disease or condition in a subject (forexample, which disease or condition can be treated or prevented with anagent which reduces the proportion of Tscm cells (e.g. which depletes ordecreases the level of said Tscm cells), or with an anti-OX40 or ananti-OX40L antibody or fragment thereof in the subject).

In one embodiment, there is provided biomarker of an autoimmune disease,HIV-1, and a T-cell malignancy, wherein the biomarker is aCD45RA+CCR7+CD95+OX40+ Tscm cell. In another embodiment, the biomarkeris of any of the diseases described in concepts 71 to 80 hereinbelow. Inanother embodiment, the Tscm cell is a CD4+CD45RA+CCR7+CD95+OX40+ Tscmcell.

Concept 41. Use of a biomarker of a Tscm-mediated disease or condition,wherein the biomarker is CD45RA+CCR7+CD95+OX40+ Tscm cells, in vitro asa diagnostic for a Tscm-mediated disease or condition (e.g. whichdisease or condition can be treated or prevented with an agent whichreduces the proportion of Tscm cells (e.g. which depletes or decreasesthe level of said Tscm cells), or with an anti-OX40 or an anti-OX40Lantibody or fragment thereof in the subject).

In one embodiment, the Tscm-mediated diseases are any of those describedin concepts 71 to 80 hereinbelow.

Concept 42. A method of maintaining CD45RA+CCR7+CD95− Tn cells, whilstdecreasing CD45RA+CCR7+CD95+OX40+ Tscm cells in a population of T-cellsin a sample, said method comprising contacting said sample with aneffective amount of an agent which reduces the proportion of Tscm cells(e.g. which depletes or decreases the level of said Tscm cells), or withan anti-OX40 or an anti-OX40L antibody or fragment thereof.

As used in concepts 1 to 83 herein, “maintains” or “maintaining” withrespect to a level or a proportion may be described as substantiallyconstant. A substantially constant level may be within 30% variancebetween samples. In one embodiment, a substantially constant level iswithin 20% variance between samples. In one embodiment, a substantiallyconstant level is within 15% variance between samples, such as within10% variance between samples, e.g. within 5% variance between samples.In another embodiment, a substantially constant level is one which doesnot show a statistically significant change in level. In one embodiment,a substantially constant level is one which reaches the 95% confidencelevel (e.g greater than 97% or greater than 99%). “Statisticallysignificant” may be as defined above in concept 28 herein.

Concept 43. A method according to concept 42, wherein the level of saidTn cells are at least maintained, whilst the levels of said Tscm cellsare decreased in said sample, optionally wherein the sample is from asubject.

Concept 44. A method according to any one of concepts 2 to 8, 10, 16,20, 21, 23 to 39, 42 or 43, an agent or an antibody or fragment thereoffor the use according to any one of concepts 12, 17, 20, 21 or 23 to 25,the use according to any one of concepts 13, 14, 18 to 20 or 23 to 25,or the composition according to concept 15, wherein agent (e.g. theantibody or fragment thereof) reduces the proportion ofCD45RA+CCR7+CD95+OX40+ Tscm cells (e.g. depletes or decreases the levelof Tscm cells).

Concept 45. A method or fragment thereof according to any one ofconcepts 1 to 8, 10, 16, 20, 21, 23 to 39 or 42 to 44, an agent or anantibody or fragment thereof for the use according to any one ofconcepts 12, 17, 20, 21, 23 to 25, or 44, the use according to any oneof concepts 13, 14, 18 to 20 or 23 to 25 or 44, or the compositionaccording to concept 15 or concept 44, wherein the agent (e.g. theantibody or fragment thereof) maintains CD45RA+CCR7+CD95− Tn cells.

Concept 46. A method according to concept 42 or 45, an agent or anantibody or fragment thereof for the use according to concept 45, theuse according to concept 45, or the composition according to concept 45,wherein the Tn cells are maintained at a level of not below 50% of thelevel of said Tn cells in a sample from a healthy donor or from saidsubject before the onset of disease.

The healthy donor is preferably of the same species at the subject, forexample, wherein the subject is a human, the donor is most preferablyalso a human. The donor is also preferably of the same gender as thesubject. The donor is preferably of a similar age and ethnicity as thesubject.

Concept 47. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 46, wherein the Tn cells aremaintained at a level of not below 55% (such as not below 60%, forexample not below 65%, e.g. not below 70%).

Concept 48. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 47, wherein the Tn cells aremaintained at a level of not below 75% (such as not below 80%, forexample not below 85%, e.g. not below 90%)

Concept 49. A method according to any one of concepts 1 to 8, or 42 to48, an agent or an antibody or fragment thereof for the use according toany one of concepts 44 to 48, the use according to any one of concepts38 to 41, or the composition according to any one of concepts 44 to 48,wherein the Tscm cells are depleted or decreased to a level of less than50% of the level of said Tscm cells in a sample from a healthy donor orfrom said subject before the onset of disease.

Concept 50. A method, an antibody or fragment for the use, a use or acomposition according to concept 49, wherein the Tscm cells are depletedor decreased to a level of less than 45% (such as less than 40%, forexample less than 35%, e.g. less than 30% or less than 25%).

Concept 51. A method, an antibody or fragment for the use, a use or acomposition according to concept 50, wherein the Tscm cells are depletedor decreased to a level of less than 20% (such as less than 15%, forexample less than 10%.

Concept 52. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody orfragment is a depleting antibody or fragment that specifically bindsOX40 (in particular human OX40), optionally wherein the antibody isengineered for enhanced ADC, ADCC and/or CDC.

The potency of Fc-mediated effects may be enhanced by engineering the Fcdomain by various established techniques. Such methods increase theaffinity for certain Fc-receptors, thus creating potential diverseprofiles of activation enhancement. This can achieved by modification ofone or several amino acid residues (e.g. as described in Lazar et al.,2006, Proc. Natl. Acad. Sci. U.S.A., March 14; 103(11):4005-10.) or byaltering the natural glycosylation profile of the Fc domain by, forexample, generating under fucosylated or de-fucosylated variants (asdescribed in Natsume et al., 2009, Drug Des Devel Ther., 3:7-16). Forexample, to increase ADCC, residues in the hinge region can be alteredto increase binding to Fc-gamma RIII (see, for example, Shields et al,2001, J Biol Chem., March 2; 276(9):6591-604.).

Equally, the enhancement of CDC may be achieved by amino acid changesthat increase affinity for C1q, the first component of the classiccomplement activation cascade (see Idusogie et al., J. Immunol., 2001;166:2571-2575). Another approach is to create a chimeric Fc domaincreated from human IgG1 and human IgG3 segments that exploit the higheraffinity if IgG3 for C1q (Natsume et al., 2008, Cancer Res., 68:3863-3872).

The antibody may be a targeting antibody (such as an anti-OX40 or ananti-OX40L antibody) which exhibits its effects through a toxin, towhich the antibody may be conjugated. Such toxins will selectively killor remove the cell to which they are targeted. Suitable immunoconjugatesare described on page 90, and on pages 114 to 118, and 134 (inparticular pages 114 to 116) herein.

Thus, in one embodiment, the antibody or fragment thereof isde-fucosylated. In another embodiment, the antibody or fragment thereofcontains one or more mutations in the hinge or Fc region which enhancesthe ADCC and/or the CDC functionality.

Methods for determining depletion and/or ADCC and/or CDC functionalitymay be as described herein, or as well-known by those skilled in theart.

The OX40 antibodies may be as described in WO2014/148895 (BioceroxProducts & Janssen Pharmaceuticals; see claims on pages 138 to 139 forspecific sequences which are incorporated herein by reference),WO2013/068563 (Biocerox Products & Janssen Pharmaceuticals; see claimson pages 138 to 139 for specific sequences which are incorporated hereinby reference), WO2013/130102 and WO2013/119202 (Providence Health &Services—Oregon, see mAb 9B12 as described in Weinberg, A. D., et al.,J. Immunother., 29, 575-585 (2006), and fusions with IL-2 which areincorporated herein by reference), WO2013/038191 (Bioceros B.V.; seeclaims 4 to 11 for specific antibody sequences which are incorporatedherein by reference), WO2013/028231 (Board of Regents, the University ofTexas System; see claims 1 to 12 for specific antibody sequences whichare incorporated herein by reference), WO2013/008171 (GlenmarkPharmaceuticals S.A.; see claims 1, 2, 5 to 12, 16 to 21 and 28 to 29for specific antibody sequences which are incorporated herein byreference), WO2012/027328 (Board of Regents, the University of TexasSystem; see claims 1 to 11 for specific antibody sequences which areincorporated herein by reference), WO2010/096418 (UCB Pharma S.A.; seeclaims 1 to 9 and 11 to 14 for specific antibody sequences which areincorporated herein by reference), WO2009/079335 (Medarex, Inc & Pfizer,Inc; see claims 1 to 9 and 14 to 17 for specific antibody sequenceswhich are incorporated herein by reference), WO2008/106116 (Genentech,Inc; see claims 1 to 12 for specific antibody sequences which areincorporated herein by reference), WO2007/062245 (Kirin Beer KabushikiKaisha & La Jolla Institute for Allergy and Immunology; see claim 12 forspecific antibody deposit numbers, and claim 16 for specific antibodysequences, which are incorporated herein by reference) WO03/106498(Crucell Holland B.V.; sec claims 3 and 4 for specific antibodysequences which are incorporated herein by reference).

More generally anti-OX40 antibodies are described in WO99/42585,WO95/21251, WO95/21915 and WO95/12673.

Concept 53. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody isan antagonistic or blocking antibody.

Methods for determining antagonism or blocking functionality may be asdescribed herein, or as well-known by those skilled in the art. Forexample, in vitro techniques include SPR and/or ELISA, which aredescribed elsewhere herein.

Concept 54. A method, an antibody or fragment for the use, a use or acomposition according to concept 53, wherein the antibody specificallybinds to OX40L (in particular human OX40L).

The OX40L antibodies may be any antibody or fragment as describedherein. In one embodiment, the OX40L antibody is the antagonistanti-human OX40L (gp34) antibody ik-1 described by Matsumura et al., JImmunol. (1999), 163:3007.

Concept 55. A method, an antibody or fragment for the use, a use or acomposition according to concept 56, wherein the antibody antagonisesspecific binding of OX40 to OX40L, e.g. as determined using SPR orELISA.

SPR and ELISA methods may be as described elsewhere herein.

Concept 56. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody isa humanized, human or fully human antibody.

Other antibody constructs may be as described herein.

Concept 57. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody isa fragment of an antibody selected from the list of multispecificantibodies (eg. bi-specific antibodies), intrabodies, single-chain Fvantibodies (scFv), camelized antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments thereof.

Concept 58. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody orfragment enables greater than 80% stem cell donor chimerism by day 12 ina Rhesus macaque model of haploidentical hematopoietic stem celltransplantation.

Concept 59. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody orfragment expresses as a stably transfected pool in Lonza GS-Xceed™ atlevel greater than 1.5 g/L in a fed batch overgrow culture using Lonzaversion 8 feed system with an overgrow period of 14 days.

Concept 60. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody orfragment thereof comprises a HCDR3 of from 16 to 27 amino acids andderived from the recombination of a human VH gene segment, a human Dgene segment and a human JH gene segment, wherein the human JH genesegment is 1GHJ6 (e.g. IGHJ6*02).

Concept 61. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody orfragment thereof comprises a HCDR3 selected from:

-   -   a. the HCDR3 of antibody 2D10 (Seq ID No:40 or Seq ID No:46);    -   b. the HCDR3 of antibody 10A7 (Seq ID No:8 or SEQ ID No:14);    -   c. the HCDR3 of antibody 09H04 (Seq ID No:72 or Seq ID No:78);    -   d. the HCDR3 of antibody 19H01 (Seq ID No:100 or Seq ID No:106);    -   e. a CDR3 of any of the nanobodies disclosed in WO2011/073180        (Ablynx, Seq ID Nos: 161 to 167 therein, which are incorporated        herein by reference);    -   f. an HCDR3 of any of the antibodies disclosed in WO2006/029879        (Roche/Genentech, Seq ID Nos: 33 to 38 therein, which are        incorporated herein by reference); or    -   g. an HCDR3 of any of the antibodies disclosed in U.S. Pat. No.        7,812,133 (Genentech, Seq ID Nos: 11 or 12 therein, which are        incorporated herein by reference).

Concept 62. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody orfragment thereof comprises:

-   -   a. the CDRs of antibody 2D10 (Seq ID No:40 or Seq ID No:46 for        CDRH3, SEQ ID No:38 or SEQ ID No:44 for CDRH2, SEQ ID No:36 or        SEQ ID No:42 for CDRH1, SEQ ID No:50 or SEQ ID No:56 for CDRL1,        SEQ ID No:52 or SEQ ID No:58 for CDRL2 and SEQ ID No:54 or SEQ        ID No:60 for CDRL3);    -   b. the CDRs of antibody 10A7 (Seq ID No:8 or SEQ ID No:14 for        CDRH3, SEQ ID No:6 or SEQ ID No:12 for CDRH2, SEQ ID No:4 or SEQ        ID No:10 for CDRH1, SEQ ID No:18 or SEQ ID No:24 for CDRL1, SEQ        ID No:20 or SEQ ID No:26 for CDRL2 and SEQ ID No:22 or SEQ ID        No:28 for CDRL3);    -   c. the CDRs of antibody 09H04 (Seq ID No:72 or Seq ID No:78 for        CDRH3, SEQ ID No:70 or SEQ ID No:76 for CDRH2, SEQ ID No:68 or        SEQ ID No:74 for CDRH1, SEQ ID No:82 or SEQ ID No:88 for CDRL1,        SEQ ID No:84 or SEQ ID No:90 for CDRL2 and SEQ ID No:86 or SEQ        ID No:92 for CDRL3);    -   d. the CDRs of antibody 19H01 (Seq ID No:100 or Seq ID No:106        for CDRH3, SEQ ID No:98 or SEQ ID No:104 for CDRH2, SEQ ID No:96        or SEQ ID No:102 for CDRH1, SEQ ID No:110 or SEQ ID No:116 for        CDRL1, SEQ ID No:112 or SEQ ID No:118 for CDRL2 and SEQ ID        No:114 or SEQ TD No:120 for CDRL3);    -   c. the CDRs of any of the nanobodies disclosed in WO2011/073180        (Ablynx: Seq ID Nos: 161 to 167 therein for CDR3; Seq ID Nos:        147 to 153 therein for CDR2; and Seq ID Nos: 133 to 139 therein        for CDR1, which sequences are incorporated herein by reference);    -   f. the CDRs of any of the antibodies disclosed in WO2006/029879        (Roche/Genentech: Seq ID Nos: 33 to 38 therein for CDRH3; Seq ID        Nos: 21 to 25 therein for CDRH1 and Seq ID Nos: 26 to 32 therein        for CDRH2; SEQ ID NOs: 39 to 44 therein for CDRL1; SEQ ID NOs:        45 to 50 therein for CDRL2; and SEQ ID NOs: 51 to 57 therein for        CDRL3, which sequences are incorporated herein by reference); or    -   g. the CDRs of any of the antibodies disclosed in U.S. Pat. No.        7,812,133 (Genentech: Seq ID Nos: 11 or 12 therein for CDRH3;        Seq ID Nos: 7 or 8 therein for CDRH1 and Seq ID Nos: 9 or 10        therein for CDRH2; SEQ ID NOs: 1 or 2 therein for CDRL1; SEQ ID        NOs: 3 or 4 therein for CDRL2; and SEQ ID NOs: 5 or 6 therein        for CDRL3, which sequences are incorporated herein by        reference).

Concept 63. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody orfragment thereof comprises the VH and/or VL domains selected from thefollowing:

-   -   a. the VH and/or VL domains of antibody 2D10 (Seq ID No:34 for        VH and/or Seq ID No:48 for VL);    -   b. the VH and/or VL domains of antibody 10A7 (Seq ID No:2 for VH        and/or Seq ID No:16 for VL);    -   c. the VH and/or VL domains of antibody 09H04 (Seq ID No:66 for        VH and/or Seq ID No:80 for VL);    -   d. the VH and/or VL domains of antibody 19H01 (Seq ID No:94 for        VH and/or Seq ID No:108 for VL);    -   e. a VH domains of any of the nanobodies disclosed in        WO2011/073180 (Ablynx, Seq ID Nos: 177 to 185, 199 to 226        therein, which sequences are incorporated herein by reference        [reproduced herein as Seq ID Nos: 177 to 213]);    -   f. the VH and/or VL domains of any of the antibodies disclosed        in WO2006/029879 (Roche/Genentech, Seq ID Nos: 2, 4, 6, 8, 10,        12, 17, 19 and 20 therein for VH domains; and Seq ID Nos: 1, 3,        5, 7, 9, 11, 16 and 18 therein for VL domains, which sequences        are incorporated herein by reference [reproduced herein as Seq        ID Nos: 214 to 230]); or    -   g. the VH and/or VL domains of any of the antibodies disclosed        in U.S. Pat. No. 7,812,133 (Genentech, Seq ID Nos: 15 and 16        therein for VH domains; and Seq ID Nos: 13 and 14 therein for VL        domains, which sequences are incorporated herein by reference        [reproduced herein as Seq ID Nos: 231 to 234]).

Concept 64. A method, an antibody or fragment for the use, a use or acomposition according to any preceding concept, wherein the antibody isoxelumab.

Concept 65. A method, an agent or an antibody or fragment for the use, ause or a composition according to any preceding concept, where in theTscm cells and/or the Tn cells are CD4+.

In another embodiment, the Tscm cells and/or the Tn cells are CD8+.

Concept 66. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 64, wherein the Tscm cellsand/or the Tn cells are circulating T-cells.

Concept 67. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 65, wherein the Tscm cellsand/or the Tn cells are in a sample of blood, e.g. peripheral blood.

Whereas T-cells present in blood are relatively straightforward toisolate and characterise, T-cells which are present in the tissues of asubject are generally more difficult to isolate. That said, it may bepossible to isolate T-cells from various tissues (such as skin, tissuesof the GI tract, e.g. bowel, and from inflamed joints, e.g. synovium)

Concept 68. A method according to any one of concepts 9 to 11, 16, 20 to39 or 43 to 67, an agent or an antibody or fragment for the useaccording to any one of concepts 12, 17, 20, 21, 23 to 25 or 44 to 67, ause according to any one of concepts 13, 14, 18 to 20 or 23 to 25 or 44to 67, or a composition according to any one of concepts 15 or 44 to 67,wherein the subject is a human patient.

Concept 69. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 68, wherein the subject is atrisk of a Tscm-mediated disease or condition.

Concept 70. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 68, wherein the subject has aTscm-mediated disease or condition.

Concept 71. A method, an agent or an antibody or fragment for the use, ause or a composition according to any preceding concept, wherein theTscm-mediated disease or condition is mediated by CD45RA+CCR7+CD95+OX40+Tscm cells.

Concept 72. A method, an agent or an antibody or fragment for the use, ause or a composition according to any one of concept 68 to 70, whereinthe Tscm-mediated disease or condition is characterised by having aratio of CD45RA+CCR7+CD95+OX40+ Tscm cells:CD45RA+CCR7+CD95− Tn cells ofgreater than 50:50.

In another embodiment, the disease or condition is characterised byhaving a ratio of Tscm cells:Tn cells as set out in any of concepts 23to 25.

Concept 73. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 72, wherein the Tscm-mediateddisease or condition is characterised by having a ratio of Tscm:Tn ofgreater than 60:40, or greater than 70:30, or greater than 75:25, suchas greater than 70:30.

Concept 74. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 73, wherein the Tscm-mediateddisease or condition is characterised by having a ratio of Tscm:Tn ofgreater than 80:20, or greater than 85:15, for example greater than90:10, e.g. greater than 95:5.

Concept 75. A method, an agent or an antibody or fragment for the use, ause or a composition according to any preceding concept, wherein theTscm-mediated disease or condition is selected from an autoimmunedisease, HIV-1, and a T-cell malignancy.

Concept 76. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 75, wherein the Tscm-mediateddisease or condition is selected from GvHD, transplant rejection,psoriasis, rheumatoid arthritis, systemic lupus erythematosus, multiplesclerosis, juvenile dermatomyositis, T-cell lymphoma and T-cellleukemia.

Concept 77. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 76, wherein the Tscm-mediateddisease or condition is GvHD or transplant rejection.

Concept 78. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 76, wherein the transplant isa cell, tissue or organ transplant (e.g. liver, lung, heart, kidney orbowel), or a blood transplant (e.g. autologous or allogeneic), forexample where the blood is bone marrow-derived, is cord-blood derived(umbilical), or is peripheral-blood derived.

In one embodiment, the transplant is a CAR T-cell transplant (chimericantigen receptor).

Concept 79. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 76, wherein the Tscm-mediateddisease or condition is a T-cell lymphoma selected from T-cellnon-Hodgkin's lymphoma, peripheral T-cell lymphoma (PTCL), anaplasticlarge cell lymphoma (ALCL), angioimmunoblastic lymphoma, cutaneousT-cell lymphoma, adult T-cell leukemia/lymphoma, blastic NK-celllymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-deltaT-cell lymphoma, lymphoblastic lymphoma (T-LBL) and nasal NK/T-celllymphoma.

Concept 80. A method, an agent or an antibody or fragment for the use, ause or a composition according to concept 76, wherein the Tscm-mediateddisease or condition is a T-cell leukemia selected from large granularlymphocytic leukemia (LGLL), T-cell prolymphocytic leukemia (T-PLL),T-cell acute lymphoblastic leukemia (T-ALL) and Sezary syndrome.

Concept 81. A method, an agent or an antibody or fragment for the use, ause or a composition according to any preceding concept, furthercomprising administering to the human a further therapeutic agent,optionally wherein the further therapeutic agent is independentlyselected from the group consisting of rapamycin (sirolimus), tacrolimus,ciclosporin, corticosteroids (e.g. methylprednisolone), methotrexate,mycophenolate mofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies(e.g. ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30antibodies (e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5receptor antagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat, in particular rapamycin(sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, CTLA4-Fc molecules (e.g. abatacept), anti-CD40L antibodies,anti-LFA1 antibodies, anti-CD52 antibodies (e.g. alemtuzumab),cyclophosphamide and anti-thymocyte globulins.

In one embodiment, the further therapeutic agent is independentlyselected from the group consisting of calcineurin inhibitors (e.g.tacrolimus, ciclosporin), mTOR inhibitors (e.g. rapamycin (sirolimus)),and antiproliferative agents (e.g. mycophenolate mofetil,cyclophosphamide).

In one embodiment, the further therapeutic agent is independentlyselected from the group consisting of immunosuppressants that modulateIL-2 signalling (e.g. tacrolimus, ciclosporin, rapamycin (sirolimus),and anti-CD25 antibodies (e.g. basilixumab, daclizumab).

In one embodiment, the further therapeutic agent is rapamycin(sirolimus). In another embodiment, the further therapeutic agent istacrolimus. In another embodiment, the further therapeutic agent is acombination of tacrolimus and methotrexate. In another embodiment, thefurther therapeutic agent is ciclosporin. In another embodiment, thefurther therapeutic agent is a combination of ciclosporin andmethotrexate. In another embodiment, the further therapeutic agent iscyclophosphamide. In another embodiment, the further therapeutic agentis mycophenolate mofetil.

Concept 82. A method, an agent or an antibody or fragment for the use, ause or a composition according to any preceding concept, furthercomprising administering to the human a therapeutically effective amountof rapamycin.

Concept 83. A method, an agent or an antibody or fragment for the use, ause or a composition according to any preceding concept, furthercomprising administering to the human a therapeutically effective amountof tacrolimus.

The inventors have surprisingly found that an anti-OX40L antibody mayprovide synergistic effects when administered as part of a combinationtherapy with a further therapeutic agent. To that end, further conceptsare provided below:

Concept 101. An anti-OX40L antibody or fragment thereof for use intreating or reducing the risk of an OX40L-mediated disease or conditionin a subject in combination with a further therapeutic agentindependently selected from the group consisting of rapamycin(sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-CD20antibodies (e.g.rituximab), anti-CD30 antibodies (e.g. brentuximab),CTLA4-Fc molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies(e.g. alemtuzumab), anti-CD45 antibodies, cyclophosphamide,anti-thymocyte globulins, anti-complement C5 antibodies (e.g.eculizumab), anti-a4b7 integrin antibodies (e.g. vedolizumab), anti-IL6antibodies (e.g. tocilizumab), anti-IL2R antibodies (e.g. basilixumab),anti-CD25 antibodies (e.g. daclizumab), anti-TNFa/TNFa-Fc molecules(e.g. etanercept, adalimumab, infliximab, golimumab or certolizumabpegol) and Vorinostat.

Concept 102. Use of an anti-OX40L antibody or fragment thereof for thetreatment or prevention of an OX40L-mediated disease or condition in asubject in combination with a further therapeutic agent independentlyselected from the group consisting of rapamycin (sirolimus), tacrolimus,ciclosporin, corticosteroids (e.g. methylprednisolone), methotrexate,mycophenolate mofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies(e.g. ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30antibodies (e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5receptor antagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat.

Concept 103. Use of an anti-OX40L antibody or fragment thereof in themanufacture of a medicament for the treatment or prevention of anOX40L-mediated disease or condition in a subject in combination with afurther therapeutic agent independently selected from the groupconsisting of rapamycin (sirolimus), tacrolimus, ciclosporin,corticosteroids (e.g. methylprednisolone), methotrexate, mycophenolatemofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies (e.g.ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30 antibodies(e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5 receptorantagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat.

Concept 104. A composition comprising an anti-OX40L antibody or fragmentthereof for the treatment or prevention of an OX40L-mediated disease orcondition in a subject in combination with a further therapeutic agentindependently selected from the group consisting of rapamycin(sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-CD20antibodies (e.g.rituximab), anti-CD30 antibodies (e.g. brentuximab),CTLA4-Fc molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies(e.g. alemtuzumab), anti-CD45 antibodies, cyclophosphamide,anti-thymocyte globulins, anti-complement C5 antibodies (e.g.eculizumab), anti-a4b7 integrin antibodies (e.g. vedolizumab), anti-IL6antibodies (e.g. tocilizumab), anti-IL2R antibodies (e.g. basilixumab),anti-CD25 antibodies (e.g. daclizumab), anti-TNFa/TNFa-Fc molecules(e.g. etanercept, adalimumab, infliximab, golimumab or certolizumabpcgol) and Vorinostat.

Concept 105. A method of treating or preventing an OX40L-mediateddisease or condition in a subject comprising administering to said humana therapeutically effective amount of an anti-OX40L antibody or fragmentthereof in combination with a further therapeutic agent independentlyselected from the group consisting of rapamycin (sirolimus), tacrolimus,ciclosporin, corticosteroids (e.g. methylprednisolone), methotrexate,mycophenolate mofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies(e.g. ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30antibodies (e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5receptor antagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat, wherein theOX40L-mediated disease or condition is thereby treated or prevented.

In any of the concepts herein, the combination may be used in theprevention of an OX40L-mediated disease. In any of the concepts, thecombination may be used to reduce the risk of an OX40L-mediated disease.In any of the concepts, the combination may be used in the treatment ofan OX40L-mediated disease.

A “combination” as described here may be as defined elsewhere herein,for example on page 100, on pages 105 to 107, and on pages 119 to 120.In one embodiment, the disease is rheumatoid arthritis or psoriasis, andthe further therapeutic agent is an anti-IL-17 antibody (such asbrodalumab, secukinumab and ixekizumab). Combinations may beadministered concomitantly or sequentially. Administration may be viaany of the methods disclosed herein, for example, as discussed in thesection entitled “Methods of Administration and Dosing” beginning onpage 130 herein.

As used in any of the concepts herein, the “treatment” of anOX40L-mediated disease includes the reduction of one or more symptom(s)of said OX40L-mediated disease. Treatment may be interpreted asdescribed elsewhere herein, for example on page 107, and in the sectionentitled “Kits” (beginning on page 149, in particular page 152)

Immunosuppressive drug intervention in the management of GvHD associatedwith hematopoietic stem cell transplant (HSCT) may be administered totreat patients with confirmed disease. GvHD grading may be determined asdescribed below.

In one embodiment, the administration is prophylactic to reduce the riskof an OX40L-mediated disease. As used in the concepts herein,“prevention” (or “prevent” or “preventing” and the like) of anOX40L-mediated disease includes the prevention of one or more symptom(s)of said OX40L-mediated disease. Preventing may refer to the total orpartial inhibition of the development, recurrence, onset or spread of anOX40L-mediated disease and/or symptom related thereto, resulting fromthe administration combination of therapies provided herein (e.g., acombination of prophylactic and/or therapeutic agents). Preventing maybe interpreted as disclosed elsewhere herein.

Immunosuppressive drug intervention in the management of GvHD associatedwith hematopoietic stem cell transplant (HSCT) may be administered toprevent disease in patients known to be at risk.

Thus, in one embodiment, a prophylactically-effective dose isadministered before the onset of an OX40L-mediated disease or condition.As used in the concepts herein, a subject may be determined to be“before the onset of an OX40L-mediated disease or condition” if thesubject is presenting no symptoms which would conventionally beassociated with said disease or condition or if the subject would not bediagnosed as having such a disease or condition by any conventionalmethod. In another embodiment, administration which is before the onertof disease may be termed “pre-emptive treatment”, which refers to theuse of further therapeutic agents (such as immunosuppressant agents)and/or an anti-OX40L antibody of the invention in individuals at risk ofdeveloping disease and where there may be early signs that emergence ofclinically-relevant GvHD is imminent. For example, an experimental orpredictive serum or cellular biomarker may indicate the optimal time forinitiation of pre-emptive GvHD treatment.

For example, the presence of signs and symptoms of acute GvHD in a humanmay be staged and graded according to a standardised scale such asdescribed in Przepiorka et al. In a primate, such as a rhesus macaquemonkey, the presence of signs and symptoms of acute GvHD may be stagedand graded according to a standardised scale such as described herein inExample 7. Similar disease grading scales are also in routine clinicaluse for other relevant diseases, such as rheumatoid arthritis andinflammatory bowel diseases.

“Prevention” or “prophylaxis” may be as described in aspect 94 herein,with dosages and timings of administration of the anti-OX40L antibody asdescribed. A prophylactic agent may be used in any of the methodsdescribed on page 102 to 103.

The OX40L-mediated diseases or conditions may be any of the diseases orconditions mentioned herein, including those which are defined elsewhereherein as Tscm-mediated diseases or conditions (see concepts 75 to 80hereinabove). In one embodiment, the OX40L-mediated diseases orconditions are as described in any of aspects 12, 12a, 69, 69a, 71, 72,72a, 90 to 93 as described herein. In one embodiment, the OX40L-mediateddiseases or conditions are as described in any of aspects 12, 12a, 69,69a, 71, 72, 72a, 90 to 93 as described herein. In one embodiment, theOX40L-mediated disease or condition is a hOX40L-mediated disease orcondition as described herein, for example on pages 103 to 104, or onpage 131. In another embodiment, the OX40L-mediated diseases orconditions are as described in the section entitled “Methods ofAdministration and Dosing” beginning on page 130 herein. In a preferredembodiment, the disease or condition is GvHD or transplant rejection, inparticular, GvHD. In another embodiment, the OX40L-mediated disease ofcondition is Crohn's disease. In another embodiment, the OX40L-mediateddisease of condition is inflammatory bowel disease (IBD). In anotherembodiment, the OX40L-mediated disease of condition is ulcerativecolitis. In another embodiment, the OX40L-mediated disease of conditionis psoriasis.

In any of concepts described herein, the anti-OX40L antibody and/or thefurther therapeutic agent are administered to the subject.Administration may be by any method described herein, for example asdescribed on page 93, or in the sections entitled “Pharmaceuticalcompositions” and “Methods of Administration” beginning on pages 118 and130 respectively. In one embodiment, the anti-OX40L antibody of theinvention is administered intravenously. In one embodiment, theanti-OX40L antibody of the invention is administered subcutaneously.

In one embodiment, the further therapeutic agent is rapamycin(sirolimus) and is administered orally. In one embodiment, the furthertherapeutic agent is tacrolimus and is administered orally. In oneembodiment, the further therapeutic agent is methotrexate and isadministered orally and/or intravenously. In one embodiment, the furthertherapeutic agent is ciclosporin and is administered intravenouslyand/or orally. In one embodiment, the further therapeutic agent iscyclophosphamide and is administered intravenously and/or orally. In oneembodiment, the further therapeutic agent is methyl prednisolone and isadministered orally and/or intravenously.

Concept 106. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any one of concepts 101to 105, wherein the subject has a post-treatment or post-prophylaxissurvival time of at least 14 days, or at least 21 days, or at least 28days, or at least 40 days, or at least 50 days, or at least 60 days.

Concept 107. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any one of concepts 101to 105, wherein post-prophylaxis, the subject has at least 7 days, or atleast 14 days, or at least 21 days, or at least 28 days, or at least 40days, or at least 50 days, or at least 60 days disease-free.

Concept 108. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any one of concepts 101to 105, wherein post-treatment, the subject has at least 7 days, or atleast 14 days, or at least 21 days, or at least 28 days, or at least 40days, or at least 50 days, or at least 60 days disease progression-free.

Concept 109. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any one of concepts 106to 108, wherein the number of days of survival, the number of diseasefree days, or the number of disease-progression free days is at least 2months, or at least 3 months, or at least 4 months, e.g. at least 5months, such as at least 6 months.

Concept 110. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 109, whereinthe number of days of survival, the number of disease free days, or thenumber of disease-progression free days is at least 9 months, or atleast one year.

Concept 111. A method of preventing the onset of an OX40L-mediateddisease or condition in a subject by administering aprophylactically-effective amount of an anti-OX40L antibody andadministering a prophylactically-effective amount of a furthertherapeutic agent independently selected from the group consisting ofrapamycin (sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-CD20antibodies (e.g.rituximab), anti-CD30 antibodies (e.g. brentuximab),CTLA4-Fc molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies(e.g. alemtuzumab), anti-CD45 antibodies, cyclophosphamide,anti-thymocyte globulins, anti-complement C5 antibodies (e.g.eculizumab), anti-a4b7 integrin antibodies (e.g. vedolizumab), anti-IL6antibodies (e.g. tocilizumab), anti-IL2R antibodies (e.g. basilixumab),anti-CD25 antibodies (e.g. daclizumab), anti-TNFa/TNFa-Fc molecules(e.g. etanercept, adalimumab, infliximab, golimumab or certolizumabpegol) and Vorinostat, wherein the onset of the OX40L-mediated diseaseor condition is prevented.

By “prophylactically-effective”, it is meant that the dose is effectiveto prevent or reduce the risk of an OX40L-mediated disease or condition.

In one embodiment, the combination may be used to reduce the risk of anOX40L-mediated disease or condition. In concept 111, the anti-OX40Lantibody of the invention and the further therapeutic agent may beadministered to the patient prophylactically, which administration maybe sequential or simultaneous. The dosing regimens and modes ofadministration may be those which are normal or traditionallyadministered by physicians for the further therapeutic agent. The dosingregimens and modes of administration may be those which are normal ortraditionally administered by physicians for the anti-OX40L antibody ofthe invention. However, the concurrent use of both agents is expected toresult in an improved prophylaxis as compared to either agent alone.

Concept 112. A method of treating an OX40L-mediated disease or conditionin a subject by administering a prophylactically-effective amount of ananti-OX40L antibody and administering a therapeutically-effective amountof a further therapeutic agent independently selected from the groupconsisting of rapamycin (sirolimus), tacrolimus, ciclosporin,corticosteroids (e.g. methylprednisolone), methotrexate, mycophenolatemofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies (e.g.ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30 antibodies(e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5 receptorantagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pcgol) and Vorinostat, wherein the onset ofthe OX40L-mediated disease or condition is treated.

By “therapeutically effective”, it is meant that the dose is effectiveto treat an OX40L-mediated disease or condition. Effective may be asdefined on pages 96 to 97, or on page 152 herein, and may provide serumconcentrations as described in the section entitled “PharmaceuticalCompositions” starting on page 118 herein.

In concept 112, the anti-OX40L antibody of the invention may beadministered to the patient, but despite prophylaxis, the onset of theOX40L-mediated disease or condition occurs (the onset may be delayed bya number of days or weeks, as compared with a patient who had not beenreceiving the antibody of the invention). In this case, atherapeutically-effective amount of a further therapeutic agent may beadministered to treat the disease or condition.

Concept 113. A method of treating an OX40L-mediated disease or conditionin a subject by administering a therapeutically-effective amount of ananti-OX40L antibody and administering a prophylactically-effectiveamount of a further therapeutic agent independently selected from thegroup consisting of rapamycin (sirolimus), tacrolimus, ciclosporin,corticosteroids (e.g. methylprednisolone), methotrexate, mycophenolatemofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies (e.g.ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30 antibodies(e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5 receptorantagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat, wherein the onset ofthe OX40L-mediated disease or condition is treated.

In concept 113, the further therapeutic agent may be administered to thepatient, but despite prophylaxis, the onset of the OX40L-mediateddisease or condition occurs (the onset may be delayed by a number ofdays or weeks, as compared with a patient who had not been receiving thefurther therapeutic agent). In this case, a therapeutically-effectiveamount of an anti-OX40L antibody of the invention may be administered totreat the disease or condition.

Concept 114. A method of treating an OX40L-mediated disease or conditionin a subject by administering a therapeutically-effective amount of ananti-OX40L antibody and administering a therapeutically-effective amountof a further therapeutic agent independently selected from the groupconsisting of rapamycin (sirolimus), tacrolimus, ciclosporin,corticosteroids (e.g. methylprednisolone), methotrexate, mycophenolatemofetil, anti-CD28 antibodies, anti-1L12/IL-23 antibodies (e.g.ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30 antibodies(e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5 receptorantagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat, wherein the onset ofthe OX40L-mediated disease or condition is treated.

In concept 114, both the anti-OX40L antibody of the invention and thefurther therapeutic agent are not admininstered until there are clinicalsigns of the OX40L-mediated disease or condition. The combinationtreatment of both agents may provide further benefits as compared toeither agent alone.

Concept 115. A method according to concept 111 or concept 113, whereinthe further therapeutic agent is independently selected from rapamycin(sirolimus), tacrolimus, a combination of tacrolimus and methotrexate,cyclophosphamide, ciclosporin, and a combination of ciclosporin andmethotrexate.

In one embodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, particularly GvHD. In one embodiment, theOX40L-mediated disease or condition is GvHD or transplant rejection,particularly GvHD, and the further therapeutic agent is rapamycin(sirolimus). In one embodiment, the OX40L-mediated disease or conditionis GvHD or transplant rejection, particularly GvHD, and the furthertherapeutic agent is tacrolimus. In one embodiment, the OX40L-mediateddisease or condition is GvHD or transplant rejection, particularly GvHD,and the further therapeutic agent is a combination of tacrolimus andmethotrexate. In one embodiment, the OX40L-mediated disease or conditionis GvHD or transplant rejection, particularly GvHD, and the furthertherapeutic agent is cyclophosphamide. In one embodiment, theOX40L-mediated disease or condition is GvHD or transplant rejection,particularly GvHD, and the further therapeutic agent is ciclosporin. Inone embodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, particularly GvHD, and the further therapeuticagent is a combination of ciclosporin and methotrexate. In oneembodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, particularly GvHD, and the further therapeuticagent is mycophenolate mofetil.

In one embodiment, the anti-OX40L antibody is administered to a patientwho is already receiving rapamycin (sirolimus). In another embodiment,embodiment, the anti-OX40L antibody is administered to a patient who isalready receiving tacrolimus. In another embodiment, the anti-OX40Lantibody is administered to a patient who is already receiving acombination of tacrolimus and methotrexate. In another embodiment, theanti-OX40L antibody is administered to a patient who is alreadyreceiving a combination of ciclosporin and methotrexate. In anotherembodiment, the anti-OX40L antibody is administered to a patient who isalready receiving ciclosporin. In another embodiment, the anti-OX40Lantibody is administered to a patient who is already receivingcyclophosphamide. In another embodiment, the anti-OX40L antibody isadministered to a patient who is already receiving mycophenolatemofetil.

These further therapeutic agents may be used prophylactically in thetreatment of OX40L-mediated diseases or conditions. For example, inGvHD, preventive therapy (prophylaxis) is typically administered aroundthe time of HSCT and is continued for a period of time followingtransplant to maintain immunosuppression during the period of greatestrisk of developing acute GvHD. Specific drug regimens differ betweentransplant centres, but as an example prophylaxis with calcineurininhibitors such as ciclosporin or tacrolimus, or with rapamycin(sirolimus) may be initiated within the 7 day period precedingtransplant (such as Day −3, or Day −1 pre-HSCT), or immediatelyfollowing the HSCT procedure (e.g. on Day 0, or Day +1 aftertransplant). Prophylaxis with mycophenolate mofetil is typically dosedfollowing HSCT, for example starting between Day +1 to Day +5post-transplant. Prophylaxis with these agents may be continued, forexample, between 28 to 180 days or longer following transplant, withdaily dosages calculated to maintain serum levels in the range toachieve effective immunosuppression without limiting side effects. Inaddition to calcineurin inhibitors, methotrexate is often used as anadjunct to prophylaxis, typically being administered on Days +1, +3, +6,and +11 post-transplant.

An anti-OX40L antibody of the invention may be used as prophylaxis incombination with tacrolimus, ciclosporin, a combination of tacrolimusand methotrexate, a combination of ciclosporin and methotrexate,cyclophosphamide, mycophenolate mofetil or rapamycin, where prophylaxiswith any of these agents is started before or around the time of HSCT,or immediately following the transplant procedure, for example withinthe period 7 days before, to 7 days after transplant. Tacrolimus, orciclosporin, or rapamycin may then be administered at therapeuticallyeffective dose and frequency, for example daily, for up to 180 daysfollowing the transplant. An anti-OX40L antibody of the invention may beadministered concurrently, starting before or around the time of HSCT,or immediately following the transplant procedure, for example withinthe period 7 days before, to 7 days after transplant. The anti-OX40Lantibody may then be dosed at a therapeutically effective dose andfrequency, for example biweekly or monthly, for up to 180 days followingthe transplant. Under these circumstances the combined activity of theanti-OX40L antibody and the additional prophylactic agent would beexpected to display a synergistic effect in preventing the onset and/orseverity of GvHD.

Concept 116. A method according to concept 112 or concept 114, whereinthe further therapeutic agent is a corticosteroid (e.g.methylprednisolone).

In one embodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD. In one embodiment, theOX40L-mediated disease or condition is GvHD or transplant rejection, inparticular GvHD, and the further therapeutic agent ismethylprednisolone.

In cases where breakthrough GvHD occurs (for example, even despiteprophylaxis), treatment may be initiated immediately upon confirmationof Grade II or higher GvHD disease. Systemic corticosteroids such asmethylprednisolone are the first-line treatment of choice, administeredconcurrently with ongoing prophylaxis with, for example, a calcineurininhibitor such as ciclosporin or tacrolimus.

Where first-line treatment or prophylaxis fails to control GvHD,“salvage therapy” may be attempted in which case additional previouslyunused immunosuppressants such as rapamycin or mycophenolate mofetil maybe administered. Thus, in one embodiment, a therapeutically effectiveamount of an anti-OX40L antibody of the invention is administered to apatient who is refractory to prophylaxis or treatment with any of:rapamycin, tacrolimus, tacrolimus in combination with methotrexate,cyclophosphamide, ciclosporin, ciclosporin in combination withmethotrexate, or corticosteroids (e.g. methylprednisolone). In anotherembodiment, a therapeutically effective amount of an anti-OX40L antibodyof the invention is administered to a patient who is refractory toprophylaxis or treatment with any of the further therapeutic agentsmentioned herein. In another embodiment, an anti-OX40L antibody of theinvention is administered as a salvage therapy.

Concept 117. A method of prolonging survival in a subject having or atrisk of an OX40L-mediated disease or condition by administering atherapeutic or prophylactic combination of an anti-OX40L antibody and afurther therapeutic agent independently selected from the groupconsisting of rapamycin (sirolimus), tacrolimus, ciclosporin,corticosteroids (e.g. methylprednisolone), methotrexate, mycophenolatemofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies (e.g.ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30 antibodies(e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5 receptorantagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat.

In one embodiment, then OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD. A number of factors affect theseverity and disease progression in GvHD, such as the pre-transplantconditioning regimen (e.g. myeloablation by irradiation, preparativechemotherapy), degree of donor-recipient tissue matching (HLA matching,and/or relationship of donor-recipient), and therapeutic or prophylactictreatment regimens.

In general, however, in primates, such as rhesus macaque that havereceived haploidential stem cell transplants, the mean survival time(MST) post-transplant and in the absence of therapy is 8 days.

Concept 118. A method according to concept 117, wherein survival isincreased by at least 7 days, or by at least 14 days, or by at least 20days, or by at least 30 days or by at least 40 days, or by at least 50days, or by at least 60 days, or by at least 70 days.

Concept 119. A method of increasing the number of disease-free, ordisease-progression free, days in a subject having or at risk of anOX40L-mediated disease or condition by administering a therapeutic orprophylactic combination of an anti-OX40L antibody and a furthertherapeutic agent independently selected from the group consisting ofrapamycin (sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-CD20antibodies (e.g.rituximab), anti-CD30 antibodies (e.g. brentuximab),CTLA4-Fc molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies(e.g. alemtuzumab), anti-CD45 antibodies, cyclophosphamide,anti-thymocyte globulins, anti-complement C5 antibodies (e.g.eculizumab), anti-a4b7 integrin antibodies (e.g. vedolizumab), anti-IL6antibodies (e.g. tocilizumab), anti-IL2R antibodies (e.g. basilixumab),anti-CD25 antibodies (e.g. daclizumab), anti-TNFa/TNFa-Fc molecules(e.g. etanercept, adalimumab, infliximab, golimumab or certolizumabpegol) and Vorinostat.

In one embodiment, then OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD. In humans, the presence ofsigns and symptoms of acute GvHD may be staged and graded according to astandardised scale such as described in Przepiorka et al. In primates,such as rhesus macaques, the presence of signs and symptoms of acuteGvHD may be staged and graded according to a standardised scale such asdescribed herein in Example 7.

Thus, the number of disease free days may be measured by absence ofclinical grading symptoms. The number of disease-progression free daysmay be measures as the number of days where the clinical grading scoredoes not change.

Concept 120. A method according to concept 119, wherein the number ofdisease-free, or disease-progression free, days is at least 7 days, orat least 14 days, or at least 21 days, or at least 28 or at least 40days, or at least 50 days, or by at least 60 days, or at least 70 days.

Concept 121. A method according to concept 120, wherein the number ofdisease free, or disease-progression free, days is at least 90 days, atleast 180 days or at least 365 days.

Concept 122. A method of treating or reducing the risk of transplantrejection or GvHD in a subject by administering a therapeutic orprophylactic combination of an anti-OX40L antibody and a furthertherapeutic agent independently selected from the group consisting ofrapamycin (sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-CD20antibodies (e.g.rituximab), anti-CD30 antibodies (e.g. brentuximab),CTLA4-Fc molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies(e.g. alemtuzumab), anti-CD45 antibodies, cyclophosphamide,anti-thymocyte globulins, anti-complement C5 antibodies (e.g.eculizumab), anti-a4b7 integrin antibodies (e.g. vedolizumab), anti-IL6antibodies (e.g. tocilizumab), anti-IL2R antibodies (e.g. basilixumab),anti-CD25 antibodies (e.g. daclizumab), anti-TNFa/TNFa-Fc molecules(e.g. etanercept, adalimumab, infliximab, golimumab or certolizumabpegol) and Vorinostat, wherein the combination results in an increasedsurvival in a Rhesus macaque model of haploidentical hematopoietic stemcell transplantation as compared to either the antibody or the furthertherapeutic agent as a monotherapy.

In one embodiment, the method is a method of preventing anOX40L-mediated disease or condition.

In one embodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD. In one embodiment, theOX40L-mediated disease or condition is GvHD or transplant rejection, inparticular GvHD, and the further therapeutic agent is a rapamycin(sirolimus). In one embodiment, the OX40L-mediated disease or conditionis GvHD or transplant rejection, in particular GvHD, and the furthertherapeutic agent is tacrolimus. In one embodiment, the OX40L-mediateddisease or condition is GvHD or transplant rejection, in particularGvHD, and the further therapeutic agent is a combination of tacrolimusand methotrexate. In one embodiment, the OX40L-mediated disease orcondition is GvHD or transplant rejection, in particular GvHD, and thefurther therapeutic agent is cyclophosphamide. In one embodiment, theOX40L-mediated disease or condition is GvHD or transplant rejection, inparticular GvHD, and the further therapeutic agent is ciclosporin. Inone embodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD, and the further therapeuticagent is a combination of ciclosporin and methotrexate. In oneembodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD, and the further therapeuticagent is mycophenolate mofetil.

The Rhesus macaque model of haploidentical hematopoietic stem celltransplantation may be as described in aspect 99 herein.

Concept 123. A method according to any one of concepts 106, 109, 110,117, 119, or 122, wherein the survival is increased by at least 7 days,or by at least 14 days, or by at least 21 days, or by at least 28 days,or by at least 40 days, or by at least 50 days, or by at least 60 days,or by at least 70 days as compared to either the antibody or the furthertherapeutic agent as a monotherapy.

Concept 124. A method according to any one of concepts 106, 109, 110,117, 119, or 122, wherein survival is at least doubled, e.g tripled, ascompared to either the antibody or the further therapeutic agent as amonotherapy.

Concept 125. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceeding concept,wherein the antibody specifically binds to human OX40L (hOX40L).

The hOX40L may be as described in aspect 28 or aspect 30 describedherein.

In any of the concepts provided herein, the anti-OX40L antibody may beas described elsewhere herein. In one embodiment, the anti-OX40Lantibody is as described in any of aspects 1 to 11, 13 to 27, 29, 31 to43 or 45 described herein. In another embodiment, the anti-OX40Lantibody is as described in aspects 73 to 89, or as in any of aspects 95to 102 described herein. In another embodiment, the anti-OX40L antibodyis as described in any of concepts 53 to 64 hereinabove. Otherproperties of anti-OX40L antibodies are described on pages 86 to 89, inthe section entitled “bispecifics” beginning on page 90 herein, in thesection entitled “Antibodies” beginning on page 108 herein and in thesection entitled “Methods of Administration and Dosing” beginning onpage 130 herein.

Concept 126. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 125, whichcompetes for binding to said hOX40L with an antibody selected from thegroup consisting of 02D10, 10A07, 09H04 and 19H01.

Competition between antibodies may be determined as described in aspect13 or aspect 73, for example as determined by SPR, ELISA, HTRF or FACS.Methods related to the measurement methods are disclosed herein,including in the Examples.

Concept 127. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 126, whichcompetes for binding to said hOX40L with the antibody 02D10, wherein theantibody or fragment comprises a VH domain which comprises a HCDR3comprising the motif VRGXYYY, wherein X is any amino acid.

Concept 128. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceeding concept,wherein the antibody antagonises specific binding of OX40 to OX40L, e.g.as determined using SPR or ELISA.

Antagonism and inhibition may be carried out as defined on page 94 to95.

Concept 129. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody is a humanized, human or fully human antibody.

Concept 130. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody is a fragment of an antibody selected from the listof multispecific antibodies (eg. bi-specific antibodies), intrabodies,single-chain Fv antibodies (scFv), camelized antibodies, Fab fragments,F(ab′) fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments thereof.

Concept 131. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody or fragment enables greater than 80% stem celldonor chimerism by day 12 in a Rhesus macaque model of haploidenticalhematopoietic stem cell transplantation.

Concept 132. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody or fragment expresses as a stably transfected poolin Lonza GS-Xceed™ at level greater than 1.5 g/L in a fed batch overgrowculture using Lonza version 8 feed system with an overgrow period of 14days.

Concept 133. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody or fragment thereof comprises a HCDR3 of from 16 to27 amino acids and derived from the recombination of a human VH genesegment, a human D gene segment and a human JH gene segment, wherein thehuman JH gene segment is 1GHJ6 (e.g. IGHJ6*02).

Concept 134. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody or fragment thereof comprises a CDR selected from:

-   -   a. the HCDR3 of antibody 2D10 (Seq ID No:40 or Seq ID No:46);    -   b. the HCDR3 of antibody 10A7 (Seq ID No:8 or SEQ ID No:14);    -   c. the HCDR3 of antibody 09H04 (Seq ID No:72 or Seq ID No:78);    -   d. the HCDR3 of antibody 19H01 (Seq ID No:100 or Seq ID No:106);    -   e. a CDR3 of any of the nanobodies having the variable region        amino acid sequence of Seq ID Nos: 177 to 213;    -   f. an HCDR3 of any of the antibodies having the variable region        amino acid sequence of Seq ID Nos: 215, 217, 219, 221, 223, 225,        227, 229 or 230; or    -   g. an HCDR3 of any of the antibodies having the variable region        amino acid sequence of Seq ID Nos: 232 or 234.

Concept 135. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody or fragment thereof comprises:

-   -   a. the CDRs of antibody 2D10 (Seq ID No:40 or Seq ID No:46 for        CDRH3, SEQ ID No:38 or SEQ ID No:44 for CDRH2, SEQ ID No:36 or        SEQ ID No:42 for CDRH1, SEQ ID No:50 or SEQ ID No:56 for CDRL1,        SEQ ID No:52 or SEQ ID No:58 for CDRL2 and SEQ ID No:54 or SEQ        ID No:60 for CDRL3);    -   b. the CDRs of antibody 10A7 (Seq ID No:8 or SEQ ID No:14 for        CDRH3, SEQ ID No:6 or SEQ ID No:12 for CDRH2, SEQ ID No:4 or SEQ        ID No:10 for CDRH1, SEQ ID No:18 or SEQ ID No:24 for CDRL1, SEQ        ID No:20 or SEQ ID No:26 for CDRL2 and SEQ ID No:22 or SEQ ID        No:28 for CDRL3);    -   c. the CDRs of antibody 09H04 (Seq ID No:72 or Seq ID No:78 for        CDRH3, SEQ ID No:70 or SEQ ID No:76 for CDRH2, SEQ ID No:68 or        SEQ ID No:74 for CDRH1, SEQ ID No:82 or SEQ ID No:88 for CDRL1,        SEQ ID No:84 or SEQ ID No:90 for CDRL2 and SEQ ID No:86 or SEQ        ID No:92 for CDRL3);    -   d. the CDRs of antibody 19H01 (Seq ID No:100 or Seq ID No:106        for CDRH3, SEQ ID No:98 or SEQ ID No:104 for CDRH2, SEQ ID No:96        or SEQ ID No:102 for CDRH1, SEQ ID No:110 or SEQ ID No:116 for        CDRL1, SEQ ID No:112 or SEQ ID No:118 for CDRL2 and SEQ ID        No:114 or SEQ TD No:120 for CDRL3);    -   e. the CDRs of any of the nanobodies having the variable region        amino acid sequence of Seq ID Nos: 177 to 213;    -   f. the heavy chain CDRs of any of the antibodies having the        heavy chain variable region amino acid sequence of Seq ID Nos:        215, 217, 219, 221, 223, 225, 227, 229 or 230, and the light        chain CDRs of any of the antibodies having the light chain        variable region amino acid sequence of Seq ID Nos: 214, 216,        218, 220, 222, 224, 226 or 228; or    -   g. the heavy chain CDRs of any of the antibodies having the        heavy chain variable region amino acid sequence of Seq ID Nos:        232 or 234, and the light chain CDRs of any of the antibodies        having the light chain variable region amino acid sequence of        Seq ID Nos:231 or 233.

Concept 136. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody or fragment thereof comprises the VH and/or VLdomains selected from the following:

-   -   a. the VH and/or VL domains of antibody 2D10 (Seq ID No:34 for        VH and/or Seq ID No:48 for VL);    -   b. the VH and/or VL domains of antibody 10A7 (Seq ID No:2 for VH        and/or Seq ID No:16 for VL);    -   c. the VH and/or VL domains of antibody 09H04 (Seq ID No:66 for        VH and/or Seq ID No:80 for VL);    -   d. the VH and/or VL domains of antibody 19H01 (Seq ID No:94 for        VH and/or Seq ID No:108 for VL);    -   e. a VH domain of any of the nanobodies having the variable        region amino acid sequence of Seq ID Nos: 177 to 213;    -   f. a VH domain of any of the antibodies having the heavy chain        variable region amino acid sequence of Seq ID Nos: 215, 217,        219, 221, 223, 225, 227, 229 or 230, and a VL domain of any of        the antibodies having the light chain variable region amino acid        sequence of Seq ID Nos: 214, 216, 218, 220, 222, 224, 226 or        228; or    -   g. a VH domain of any of the antibodies having the heavy chain        variable region amino acid sequence of Seq ID Nos: 232 or 234,        and a VL domain of any of the antibodies having the light chain        variable region amino acid sequence of Seq ID Nos:231 or 233.

Concept 137. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the antibody is oxelumab.

Concept 138. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the subject is a primate.

The term “subject” may be other subjects as described herein, forexample as described on page 104. In one embodiment, the primate is arhesus macaque monkey.

Concept 139. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the subject is a human.

In one embodiment, the subject is a human patient.

Concept 140. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the subject is at risk of an OX40L-mediated disease orcondition.

In one embodiment, a subject may be identified as being “at risk of anOX40L-mediated disease or condition” if the subject has been previouslyidentified as having an increased risk, e.g. by genotyping and/orphenotyping, but the subject has not yet presented symptoms or would notbe diagnosed as having such a disease or condition by any conventionalmethod. Thus, the methods and uses disclosed herein may aid in the earlyidentification of patients who will develop such diseases or conditions.In one embodiment, the disease or condition is prevented (i.e. thetreatment is prophylactic).

In one embodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD. In a particular embodiment,the subject is at risk of GvHD or transplant rejection when they arepre-operative for a transplant. In particular, a subject is at risk ofGvHD or transplant rejection when they have commenced a pre-transplantconditioning regimen (e.g. myeloablation by irradiation, preparativechemotherapy), and when degree of donor-recipient tissue matching (HLAmatching, and/or relationship of donor-recipient) is not 100%. Potentialtransplant therapies are envisaged in concept 78 hereinabove.

Concept 141. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 40, wherein themethod, antibody or fragment for the use, the use or the composition isfor the prevention of the OX40L-mediated disease or condition.

Concept 142. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any one of concepts 101to 139, wherein the subject has an OX40L-mediated disease or condition.

A subject has an OX40L-mediated disease or condition if the subject ispresenting symptoms which would conventionally be associated with saiddisease or condition or if the subject would be diagnosed as having sucha disease or condition by any conventional method. In one embodiment,the OX40L-mediated disease or condition is GvHD or transplant rejection,particularly GvHD, and a subject may be determined to have the diseaseby any of the methods mentioned in concepts 101 to 105 and 119hereinabove.

Concept 143. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 142, whereinthe method, antibody or fragment for the use, the composition for theuse, or the use is for the treatment of the OX40L-mediated disease orcondition.

In one embodiment, treatment is commenced when the OX40L-mediateddisease or condition has been diagnosed as a confirmed disease orcondition.

In one embodiment, the OX40L-mediated disease or condition is GvHD ortransplant rejection, in particular GvHD. GvHD grading may be determinedas described herein (see concepts 101 to 105 and 119 hereinabove). Inone embodiment, the subject is a human having Grade II clinical symptomsof GvHD.

Concept 144. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any preceding concept,wherein the OX40L-mediated disease or condition is selected from anautoimmunc disease or condition, a systemic inflammatory disease orcondition, or transplant rejection.

Concept 145. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 144, whereinthe OX40L-mediated disease or condition is selected from inflammatorybowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplantrejection, allogeneic transplant rejection, graft-versus-host disease(GvHD), ulcerative colitis, systemic lupus erythematosus (SLE),diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity,multiple sclerosis and atherosclerosis.

Concept 146. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 145, whereinthe OX40L-mediated disease or condition is GvHD or transplant rejection.

Concept 147. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 146, whereinthe OX40L-mediated disease or condition is GvHD.

Concept 148. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 146, whereinthe transplant is a cell, tissue or organ transplant (e.g. liver, lung,heart, kidney or bowel), or a blood transplant (e.g. autologous orallogeneic), for example where the blood is bone marrow-derived, iscord-blood derived (umbilical), or is peripheral-blood derived.

Concept 149. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any one of concepts 146to 148, wherein the anti-OX40L antibody or fragment thereof isadministered before transplant.

Concept 150. A method, an antibody or fragment for the use, acomposition for the use, or the use according to any one of concepts 146to 148, wherein the anti-OX40L antibody or fragment thereof isadministered after transplant.

Concept 151. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 149 or concept150, wherein the further therapeutic agent is administered aftertransplant.

In one embodiment, the further therapeutic agent is rapamycin(sirolimus). In one embodiment, the further therapeutic agent istacrolimus. In one embodiment, the further therapeutic agent is acombination of tacrolimus and methotrexate. In one embodiment, thefurther therapeutic agent is cyclophosphamide. In one embodiment, thefurther therapeutic agent is ciclosporin. In one embodiment, the furthertherapeutic agent is a combination of ciclosporin and methotrexate. Inone embodiment, the further therapeutic agent is mycophenolate mofetil.In one embodiment, the further therapeutic agent is methylpredinsolonel.

Concept 152. A method, an antibody or fragment for the use, acomposition for the use, or the use according to concept 149 or concept150, wherein the further therapeutic agent is administered beforetransplant.

In one embodiment, the further therapeutic agent is rapamycin(sirolimus). In one embodiment, the further therapeutic agent istacrolimus. In one embodiment, the further therapeutic agent is acombination of tacrolimus and methotrexate. In one embodiment, thefurther therapeutic agent is cyclophosphamide. In one embodiment, thefurther therapeutic agent is ciclosporin. In one embodiment, the furthertherapeutic agent is a combination of ciclosporin and methotrexate. Inone embodiment, the further therapeutic agent is mycophenolate mofetil.

Concept 153. A pharmaceutical composition comprising an anti-OX40Lantibody or fragment thereof and a pharmaceutically acceptableexcipient, diluent or carrier and further comprising a furthertherapeutic agent independently selected from the group consisting ofrapamycin (sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, anti-IL12/IL-23 antibodies (e.g. ustekinumab), anti-CD20antibodies (e.g.rituximab), anti-CD30 antibodies (e.g. brentuximab),CTLA4-Fc molecules (e.g. abatacept), CCR5 receptor antagonists (e.g.maraviroc), anti-CD40L antibodies, anti-VLA4 antibodies (e.g.natalizumab), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies(e.g. alemtuzumab), anti-CD45 antibodies, cyclophosphamide,anti-thymocyte globulins, anti-complement C5 antibodies (e.g.eculizumab), anti-a4b7 integrin antibodies (e.g. vedolizumab), anti-IL6antibodies (e.g. tocilizumab), anti-IL2R antibodies (e.g. basilixumab),anti-CD25 antibodies (e.g. daclizumab), anti-TNFa/TNFa-Fc molecules(e.g. etanercept, adalimumab, infliximab, golimumab or certolizumabpegol) and Vorinostat.

In one embodiment, there is provided a composition or kit for treatingand/or preventing a OX40L-mediated condition or disease, the compositionor kit comprising an antibody or fragment of the invention incombination with a further therapeutic agent optionally in combinationwith a label or instructions for use to treat and/or prevent saiddisease or condition in a human; optionally wherein the label orinstructions comprise a marketing authorisation number (e.g., an FDA orEMA authorisation number); optionally wherein the kit comprises an IV orinjection device that comprises the antibody or fragment.

The composition may be as described in aspect 105, 106 or 107 herein.Excipients for use in pharmaceutical formulations are well-known to theskilled person and may be as defined on page 97 herein, or in thesection entitled “Pharmaceutical Compositions” beginning on page 118herein, or in the section entitled “Methods of Administration andDosing” beginning on page 130 herein.

Concept 154. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, wherein the further therapeutic agent isindependently selected from the group consisting of rapamycin(sirolimus), tacrolimus, ciclosporin, corticosteroids (e.g.methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28antibodies, CTLA4-Fc molecules (e.g. abatacept), anti-CD40L antibodies,anti-LFA1 antibodies, anti-CD52 antibodies (e.g. alemtuzumab),cyclophosphamide and anti-thymocyte globulins.

Other combinations may be with the anti-inflammatory drugs described inaspect 46 herein, or as described in aspect 103. Other combinations areas described in concepts 81 to 83 hereinabove, or in any of concepts 101to 153 hereinabove.

Concept 155. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof a further therapeutic agent independently selected from the groupconsisting of rapamycin, tacrolimus, ciclosporin, cyclophosphamide,corticosteroids (e.g. methylprednisolone), methotrexate or mycophenolatemofetil, anti-CD28 antibodies, CTLA4-Fc molecules (e.g. abatacept) andanti-thymocyte globulins.

Concept 156. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof a further therapeutic agent independently selected from the groupconsisting of rapamycin, tacrolimus, ciclosporin, cyclophosphamide,corticosteroids (e.g. methylprednisolone), methotrexate andmycophenolate mofetil.

Concept 157. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof a further therapeutic agent independently selected from the groupconsisting of an immunosuppressant that modulate IL-2 signalling (e.g.tacrolimus, ciclosporin, rapamycin (sirolimus)), and anti-CD25antibodies (e.g. basilixumab, daclizumab).

Concept 158. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof a further therapeutic agent independently selected from the groupconsisting of calcineurin inhibitors (e.g. tacrolimus, ciclosporin),mTOR inhibitors (e.g. rapamycin (sirolimus)), and antiproliferativeagents (e.g. mycophenolate mofetil, cyclophosphamide).

Concept 159. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof rapamycin.

Concept 160. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof tacrolimus.

Concept 161. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof tacrolimus and methotrexate.

Concept 162. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof ciclosporin.

Concept 163. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof ciclosporin and methotrexate.

Concept 164. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof cyclophosphamide.

Concept 165. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof mycophenolate mofetil.

Concept 166. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, further comprising administering to the human atherapeutically effective amount, or a prophylactically effective amountof a corticosteroid (e.g. methyl prednisolone).

Concept 167. A method, an antibody or fragment for the use, acomposition for the use, the use or the composition according to anypreceding concept, wherein the further therapeutic agent is administeredsequentially or simultaneously with the anti-hOX40L antibody orfragment.

As explained in the examples, the inventors devised a set of criteriathat is particularly useful for identifying antibodies and fragments ofthe invention, these criteria being:—

-   -   (a) The ability of the antibody or fragment to bind cell-surface        hOX40L on CHO-S cells (optionally transfected with full length        human OX40L) and/or bind recombinant hOX40L in a HTRF assay;    -   (b) The ability of the antibody or fragment to neutralise human        OX40 (e.g. neutralise human OX40L binding to human OX40        Receptor) in a receptor neutralisation HTRF assay and/or a flow        cytometry receptor neutralisation assay; and    -   (c) The ability of the antibody or fragment to specifically bind        both human and rhesus monkey OX40L (useful so that the PK, PD,        efficacy and other parameters of the antibody or fragment can be        assessed in the rhesus model as a surrogate for humans).

Thus, in an example of the invention the antibody or fragment meetscriteria (a), (b) and (c).

In an example, criterion (a) is set so that the antibody or fragmentshows <70% receptor binding by FACS to hOX40L expressed by CHO-S cells.

In an example, criterion (a) is set so that the antibody or fragmentshows <90% of receptor binding to OX40L in the HTRF assay.

In an example, criterion (a) is set so that the antibody or fragmentshows at least a 20% effect in the HTRF assay.

In an example, OX40 is used in criterion (b).

In an embodiment, assaying or testing of an antibody or fragment of theinvention is carried out at or substantially at pH7 (e.g., for in vitrotests and assays) and at or substantially at rtp.

Optionally, the antibody or fragment specifically binds hOX40L with anaffinity (apparent affinity, Kd) of less than 1 microM, 1000 nM to 100nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM,less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM,10 pM to 1 pM, e.g., in the range of 1 mM to 1 pM (e.g., 1 mM to 100 pM;10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM) as determined by SPR,e.g., under SPR conditions disclosed herein). Additionally oralternatively, the antibody or fragment specifically binds rhesus monkeyOX40L with an affinity (apparent affinity, Kd) of less than 1 microM,1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM,500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM,100 pM to 10 pM, 10 pM to 1 pM, e.g., in the range of 1 mM to 1 pM(e.g., 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1pM) as determined by SPR, e.g., under SPR conditions disclosed herein).Such binding measurements can be made using a variety of binding assaysknown in the art, e.g., using surface plasmon resonance (SPR), such asby Biacore™ or using the ProteOn XPR36™ (Bio-Rad®), using KinExA®(Sapidyne Instruments, Inc), or using ForteBio Octet (Pall ForteBioCorp.).

OX40L binding ability, specificity and affinity (Kd, K_(off) and/orK_(on)) can be determined by any routine method in the art, e.g., bysurface plasmon resonance (SPR). The term “Kd”, as used herein, isintended to refer to the equilibrium dissociation constant of aparticular antibody-antigen interaction.

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (e.g., using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,e.g., 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0.05% by volume, e.g., in the presence of P20 (polysorbate20; e.g., Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (e.g., P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the antibody or fragment is determinedusing SPR by

-   1. Coupling anti-mouse (or other relevant human, rat or non-human    vertebrate antibody constant region species-matched) IgG (e.g.,    Biacore™ BR-1008-38) to a biosensor chip (e.g., GLM chip) such as by    primary amine coupling;-   2. Exposing the anti-mouse IgG (or other matched species antibody)    to a test IgG antibody to capture test antibody on the chip;-   3. Passing the test antigen over the chip's capture surface at 1024    nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and-   4. And determining the affinity of binding of test antibody to test    antigen using surface plasmon resonance, e.g., under an SPR    condition discussed above (e.g., at 25° C. in physiological buffer).    SPR can be carried out using any standard SPR apparatus, such as by    Biacore™ or using the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, e.g., using amodel inherent to the ProteOn XPR36™ analysis software.

In an example, the antibody or fragment of the invention is contained ina medical container, e.g., a vial, syringe, IV container or an injectiondevice (e.g., an intraocular or intravitreal injection device). In anexample, the antibody or fragment is in vitro, e.g., in a sterilecontainer. In an example, the invention provides a kit comprising theantibody or fragment of the invention, packaging and instructions foruse in treating or preventing or diagnosing in a human a disease orcondition mediated by the OX40L. In an example, the instructionsindicate that the human should be genotyped for an OX40L variantsequence of the invention before administering the antibody or fragmentto the human. In an example, the instructions indicate that the humanshould be phenotyped for an OX40L variant of the invention beforeadministering the antibody or fragment to the human. In an example, thehuman is of Chinese (e.g., Han or CHS) ethnicity and the instructionsare in Chinese (e.g., Mandarin).

In an example the binding site(s) of the antibody or fragment areselected from a plurality (e.g., library) of binding sites. For example,the plurality of binding sites comprises or consists of a plurality of4-chain antibodies or fragments thereof, e.g., dAbs, Fabs or scFvs.Suitable methods for producing pluralities of binding sites forscreening include phage display (producing a phage display library ofantibody binding sites), ribosome display (producing a ribosome displaylibrary of antibody binding sites), yeast display (producing a yeastdisplay library of antibody binding sites), or immunisation of anon-human vertebrate (e.g., a rodent, e.g., a mouse or rat, e.g., aVelocimouse™, Kymouse™, Xenomouse™, Aliva Mouse™, HuMab Mouse™,Omnimouse™, Omnirat™ or MeMo Mouse™) with hOX40L or a hOX40L epitope andisolation of a repertoire of antibody-producing cells (e.g., a B-cell,plasma cell or plasmablast repertoire) and/or a repertoire of isolatedantibodies, fragments or binding sites.

The term “epitope” is a region of an antigen that is bound by anantibody or fragment. Epitopes may be defined as structural orfunctional. Functional epitopes are generally a subset of the structuralepitopes and have those residues that directly contribute to theaffinity of the interaction. Epitopes may also be conformational, thatis, composed of non-linear amino acids. In certain embodiments, epitopesmay include determinants that are chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl groups, orsulfonyl groups, and, in certain embodiments, may have specificthree-dimensional structural characteristics, and/or specific chargecharacteristics.

The term “isolated” with reference to any aspect of the invention, e.g.,an antibody or fragment, means that a subject antibody or fragment etc.(1) is free of at least some other proteins with which it would normallybe found, (2) is essentially free of other proteins from the samesource, e.g., from the same species, (3) is expressed by a cell from adifferent species, (4) has been separated from at least about 50 percentof polynucleotides, lipids, carbohydrates, or other materials with whichit is associated in nature, (5) is operably associated (by covalent ornoncovalent interaction) with a polypeptide with which it is notassociated in nature, or (6) does not occur in nature. Typically, an“isolated” antibody, fragment, etc. constitutes at least about 5%, atleast about 10%, at least about 25%, or at least about 50%, 60%, 70%,80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or >99% of a given sample.Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or anycombination thereof can encode such an isolated antibody, fragment, etc.Preferably, the isolated antibody, fragment, etc. is substantially freefrom proteins or polypeptides or other contaminants that are found inits natural environment that would interfere with its therapeutic,diagnostic, prophylactic, research or other use.

For example, an “isolated” antibody is one that has been identified,separated and/or recovered from a component of its productionenvironment (e.g., naturally or recombinantly). Preferably, the isolatedpolypeptide is free of association with all other components from itsproduction environment, e.g., so that the antibody has been isolated toan FDA-approvable or approved standard. Contaminant components of itsproduction environment, such as that resulting from recombinanttransfected cells, are materials that would typically interfere withresearch, diagnostic or therapeutic uses for the antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide will be purified: (1)to greater than 95% by weight of antibody as determined by, for example,the Lowry method, and in some embodiments, to greater than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, an isolated polypeptide or antibodywill be prepared by at least one purification step.

Immunoconjugates

The invention encompasses the antibody or fragment conjugated to atherapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxinagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxin agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, see for example, WO 05/103081,which is incorporated by reference herein in its entirety.

Bispecifics

The antibodies and fragments of the present invention may bemonospecific, bispecific, or multispecific. Multispecific mAbs may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., (1991) J. Immunol. 147:60-69. The humananti-hOX40L antibodies or fragments can be linked to or co-expressedwith another functional molecule, e.g., another peptide or protein. Forexample, an antibody or fragment thereof can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody or antibody fragment, to produce a bispecific or amultispecific antibody with a second binding specificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)CH3 domain and a second Ig CH3 domain, wherein the first and second IgCH3 domains differ from one another by at least one amino acid, andwherein at least one amino acid difference reduces binding of thebispecific antibody to Protein A as compared to a bi-specific antibodylacking the amino acid difference. In one embodiment, the first Ig CH3domain binds Protein A and the second Ig CH3 domain contains a mutationthat reduces or abolishes Protein A binding such as an H95R modification(by IMGT exon numbering; H435R by EU numbering). The second CH3 mayfurther comprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N,V397M, and V4221 by EU) in the case of IgG1 antibodies; N44S, K52N, andV821 (IMGT; N384S, K392N, and V4221 by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the presentinvention.

In certain embodiments, the antibody or OX40L binding fragment thereofcomprises less than six CDRs. In some embodiments, the antibody orantigen binding fragment thereof comprises or consists of one, two,three, four, or five CDRs selected from the group consisting of HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In specific embodiments, theantibody or antigen binding fragment thereof comprises or consists ofone, two, three, four, or five CDRs selected from the group consistingof the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences in thesequence listing (i.e. Seq ID No:4, Seq ID No:10, Seq ID No:36, Seq IDNo:42, Seq ID No:68, Seq ID No:74, Seq ID No:96 or Seq ID No:102, inparticular, Seq ID No:36 or Seq ID No:42 for HCDR1; Seq ID No:6, Seq IDNo:12, Seq ID No:38, Seq ID No:44, Seq ID No:70, Seq ID No:76, Seq IDNo:98 or Seq ID No:104, in particular Seq ID No:38 or Seq ID No:44 forHCDR2; Seq ID No:8, Seq ID No:14, Seq ID No:40, Seq ID No:46, Seq IDNo:72, Seq ID No:78, Seq ID No:100 or Seq ID No:106, in particular SeqID No:40 or Seq ID No:46 for HCDR3; Seq ID No:18, Seq ID No:24, Seq IDNo:50, Seq ID No:56, Seq ID No:82, Seq ID No:88, Seq ID No:110 or Seq IDNo:116, in particular Seq ID No:50 or Seq ID No:56 for LCDR1; Seq IDNo:20, Seq ID No:26, Seq ID No:52, Seq ID No:58, Seq ID No:84, Seq IDNo:90, Seq ID No:112 or Seq ID No:118, in particular Seq ID No:52 or SeqID No:58 for LCDR2; and Seq ID No:22, Seq ID No:28, Seq ID No:54, Seq IDNo:60, Seq ID No:86, Seq ID No:92, Seq ID No:114 or Seq ID No:120, inparticular Seq ID No:54 or Seq ID No:60 for LCDR3).

In specific embodiments, an antibody of the invention is a fully humanantibody, a monoclonal antibody, a recombinant antibody, an antagonistantibody, a hOX40L-neutralising antibody or any combination thereof orthe invention provides a hOX40L binding fragment thereof. In an example,the antibody is a chimaeric antibody comprising human variable domainsand non-human (e.g., mouse or rat or rabbit) constant domains. Inparticular embodiments, the antibody is a fully human antibody, such asa fully human monoclonal antibody, or antigen binding fragment thereof,that specifically binds to hOX40L. In preferred embodiments, theantibody is an antagonist antibody. In preferred embodiments, theantibody is a neutralising antibody.

In an example, the antibody or fragment is a lambda-type antibody orfragment (i.e., whose variable domains are lambda variable domains).Optionally, the antibody or fragment also comprises lambda constantdomains.

In certain embodiments, the antibody competes (e.g., in a dose dependentmanner) with OX40 or a fusion protein thereof (e.g., Fc:OX40), forbinding to hOX40L, such as a cell surface-expressed hOX40L or solublehOX40L. Exemplary competitive blocking tests are provided in theExamples herein.

In another aspect, provided herein are isolated nucleic acids encodingantibodies that specifically bind to a hOX40L polypeptide (e.g., a cellsurface-expressed or soluble hOX40L), a hOX40L polypeptide fragment, ora hOX40L epitope. In certain embodiments, the nucleic acid encodes a VHchain, VL chain, VH domain, VL domain, HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 as disclosed in the sequence listing (i.e. Seq ID No:30or Seq ID No:62 for VH chains; Seq ID No:32 or Seq ID No:64 for VLchains; Seq ID No: Seq ID No:2, Seq ID No:34, Seq ID No:66 or Seq IDNo:94, in particular Seq ID No:34 for VH domains; Seq ID No:16, Seq IDNo:48, Seq ID No:80, or Seq ID No:108, in particular Seq ID No:48 for VLdomains; Seq ID No:4, Seq ID No:10, Seq ID No:36, Seq ID No:42, Seq IDNo:68, Seq ID No:74, Seq ID No:96 or Seq ID No:102, in particular, SeqID No:36 or Seq ID No:42 for HCDR1; Seq ID No:6, Seq ID No:12, Seq IDNo:38, Seq ID No:44, Seq ID No:70, Seq ID No:76, Seq ID No:98 or Seq IDNo:104, in particular Seq ID No:38 or Seq ID No:44 for HCDR2; Seq IDNo:8, Seq ID No:14, Seq ID No:40, Seq ID No:46, Seq ID No:72, Seq IDNo:78, Seq ID No:100 or Seq ID No:106, in particular Seq ID No:40 or SeqID No:46 for HCDR3; Seq ID No:18, Seq ID No:24, Seq ID No:50, Seq IDNo:56, Seq ID No:82, Seq ID No:88, Seq ID No:110 or Seq ID No:116, inparticular Seq ID No:50 or Seq ID No:56 for LCDR1; Seq ID No:20, Seq IDNo:26, Seq ID No:52, Seq ID No:58, Seq ID No:84, Seq ID No:90, Seq IDNo:112 or Seq ID No:118, in particular Seq ID No:52 or Seq ID No:58 forLCDR2; and Seq ID No:22, Seq ID No:28, Seq ID No:54, Seq ID No:60, SeqID No:86, Seq ID No:92, Seq ID No:114 or Seq ID No:120, in particularSeq ID No:54 or Seq ID No:60 for LCDR3).

In another aspect, provided herein are vectors and host-cells comprisingnucleic acids encoding antibodies or fragments of the invention.

In certain embodiments, the antibody specifically binds to one or moresingle nucleotide polymorphism (SNP) variants of hOX40L. In an exampleof any aspect of the invention, the hOX40L is a trimer of monomers.

In an aspect, provided herein is a method for decreasing (e.g., by atleast 20, 30, 40 50 or 60%, or 70%, 80%, 90%, 95% or >90%) or completelyinhibiting binding of hOX40L to OX40 in a subject (e.g., a humansubject), comprising administering to the subject an effective amount ofan antibody or fragment thereof of the invention that specifically bindsto hOX40L (e.g., a cell surface-expressed or soluble hOX40L).

In an aspect, provided herein is a method of treating or preventing ahOX40L-mediated disease or condition in a subject (e.g., a humansubject), the method comprising administering to the subject aneffective amount of an antibody or fragment thereof of the inventionthat specifically binds to hOX40L (e.g., a cell surface-expressed orsoluble hOX40L), wherein the disease or condition is treated orprevented by the antibody or fragment. In an example, the methodcomprises decreasing or inhibiting a hOX40L biological activity, such assecretion of one, more or all of IL-2, IL-8, TNF alpha and interferongamma, in the subject. In an example, the biological activity isselected from the secretion of one, more or all of IL-2, TNF alpha andinterferon gamma. In an example, the biological activity is selectedfrom the secretion of one, more or all of IL-8, CCL20 and RANTES.

In an aspect, provided herein is a method of decreasing or inhibiting ahOX40L biological activity, such as secretion of one, more or all ofIL-2, IL-8, TNF alpha and interferon gamma, in a subject (e.g., a humansubject), the method comprising administering to the subject aneffective amount of an antibody or fragment thereof of the inventionthat specifically binds to hOX40L (e.g., a cell surface-expressed orsoluble hOX40L), wherein hOX40L biological activity is decreased by theantibody or fragment. In an example, the biological activity is selectedfrom the secretion of one, more or all of IL-2, TNF alpha and interferongamma. In an example, the biological activity is selected from thesecretion of one, more or all of IL-8, CCL20 and RANTES.

The term “about” or “approximately” means within 20%, preferably within10%, and more preferably within 5% (or 4%, or 3% or 2%, or, in anexample, 1% or less) of a given value or range.

As used herein, “administer” or “administration” refers to the act ofinjecting or otherwise physically delivering a substance as it existsoutside the body (e.g., an anti-hOX40L antibody provided herein) into apatient, such as by mucosal, intradermal, intravenous, intramusculardelivery and/or any other method of physical delivery described hereinor known in the art. When a disease, or a symptom thereof, is beingtreated, administration of the substance typically occurs after theonset of the disease or symptoms thereof. When a disease, or symptomsthereof, are being prevented, administration of the substance typicallyoccurs before the onset of the disease or symptoms thereof.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences (e.g., aminoacid sequences or nucleic acid sequences) can also be accomplished usinga mathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci.U.S.A. 87:2264 2268, modified as in Karlin and Altschul, 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present invention. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g., to score 50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively,PSI BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,National Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov). Another preferred, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, an “antagonist” or “inhibitor” of hOX40L refers to aligand (e.g., antibody or fragment) that is capable of inhibiting orotherwise decreasing one or more of the biological activities of hOX40L,such as in a cell expressing hOX40L or in a cell expressing a hOX40Lligand. For example, in certain embodiments, antibodies of the inventionare antagonist antibodies that inhibit or otherwise decrease secretionof CCL20, IL-8 and/or RANTES from a cell having a cell surface-expressedOX40 when said antibody is contacted with said cell. In someembodiments, an antagonist of hOX40L (e.g., an antagonistic antibody ofthe invention) may, for example, act by inhibiting or otherwisedecreasing the activation and/or cell signalling pathways of the cellexpressing OX40L, thereby inhibiting a hOX40L-mediated biologicalactivity of the cell the relative to the hOX40L-mediated biologicalactivity in the absence of antagonist. In certain embodiments, theantibodies provided herein are fully human, antagonistic anti-hOX40Lantibodies, preferably fully human, monoclonal, antagonistic anti-hOX40Lantibodies.

The term “antibody” and “immunoglobulin” or “Ig” may be usedinterchangeably herein. An antibody or a fragment thereof thatspecifically binds to a hOX40L antigen may be cross-reactive withrelated antigens. Preferably, an antibody or a fragment thereof thatspecifically binds to a hOX40L antigen does not cross-react with otherantigens (but may optionally cross-react with OX40L of a differentspecies, e.g., rhesus, or murine). An antibody or a fragment thereofthat specifically binds to a hOX40L antigen can be identified, forexample, by immunoassays, BIAcore™, or other techniques known to thoseof skill in the art. An antibody or a fragment thereof bindsspecifically to a hOX40L antigen when it binds to a hOX40L antigen withhigher affinity than to any cross-reactive antigen as determined usingexperimental techniques, such as radioimmunoassays (RIA) andenzyme-linked immunosorbent assays (ELISAs). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 times background. See, e.g., Paul, ed.,1989, Fundamental Immunology Second Edition, Raven Press, New York atpages 332-336 for a discussion regarding antibody specificity.

Antibodies of the invention include, but are not limited to, syntheticantibodies, monoclonal antibodies, recombinantly produced antibodies,multispecific antibodies (including bi-specific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, intrabodies,single-chain Fvs (scFv) (e.g., including monospecific, bispecific,etc.), camelized antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. In particular, antibodiesof the present invention include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,antigen binding domains or molecules that contain an antigen-bindingsite that specifically binds to a hOX40L antigen (e.g., one or morecomplementarity determining regions (CDRs) of an anti-hOX40L antibody).The antibodies of the invention can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2, in particular IgG4), or any subclass (e.g., IgG2a and IgG2b) ofimmunoglobulin molecule. In preferred embodiments, the hOX40L antibodiesare fully human, such as fully human monoclonal hOX40L antibodies. Incertain embodiments, antibodies of the invention are IgG antibodies, ora class (e.g., human IgG1 or IgG4) or subclass thereof. In certainembodiments, the antibodies of the invention comprise a human gamma 4constant region. In another embodiment, the heavy chain constant regiondoes not bind Fc-γ receptors, and e.g. comprises a Leu235Glu mutation.In another embodiment, the heavy chain constant region comprises aSer228Pro mutation to increase stability. In another embodiment, theheavy chain constant region is IgG4-PE.

The term “antigen binding domain,” “antigen binding region,” “antigenbinding fragment,” and similar terms refer to that portion of anantibody which comprises the amino acid residues that interact with anantigen and confer on the binding agent its specificity and affinity forthe antigen (e.g., the complementarity determining regions (CDRs)). Theantigen binding region can be derived from any animal species, such asrodents (e.g., rabbit, rat or hamster) and humans. Preferably, theantigen binding region will be of human origin.

As used herein, the term “composition” is intended to encompass aproduct containing the specified ingredients (e.g., an antibody of theinvention) in, optionally, the specified amounts, as well as any productwhich results, directly or indirectly, from combination of the specifiedingredients in, optionally, the specified amounts.

In the context of a polypeptide, the term “derivative” as used hereinrefers to a polypeptide that comprises an amino acid sequence of ahOX40L polypeptide, a fragment of a hOX40L polypeptide, or an antibodythat specifically binds to a hOX40L polypeptide which has been alteredby the introduction of amino acid residue substitutions, deletions oradditions. The term “derivative” as used herein also refers to a hOX40Lpolypeptide, a fragment of a hOX40L polypeptide, or an antibody thatspecifically binds to a hOX40L polypeptide which has been chemicallymodified, e.g., by the covalent attachment of any type of molecule tothe polypeptide. For example, but not by way of limitation, a hOX40Lpolypeptide, a fragment of a hOX40L polypeptide, or a hOX40L antibodymay be chemically modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. The derivatives are modified in a mannerthat is different from naturally occurring or starting peptide orpolypeptides, either in the type or location of the molecules attached.Derivatives further include deletion of one or more chemical groupswhich are naturally present on the peptide or polypeptide. A derivativeof a hOX40L polypeptide, a fragment of a hOX40L polypeptide, or a hOX40Lantibody may be chemically modified by chemical modifications usingtechniques known to those of skill in the art, including, but notlimited to specific chemical cleavage, acetylation, formulation,metabolic synthesis of tunicamycin, etc. Further, a derivative of ahOX40L polypeptide, a fragment of a hOX40L polypeptide, or a hOX40Lantibody may contain one or more non-classical amino acids. Apolypeptide derivative possesses a similar or identical function as ahOX40L polypeptide, a fragment of a hOX40L polypeptide, or a hOX40Lantibody described herein.

The term “effective amount” as used herein refers to the amount of atherapy (e.g., an antibody or pharmaceutical composition providedherein) which is sufficient to reduce and/or ameliorate the severityand/or duration of a given disease and/or a symptom related thereto.This term also encompasses an amount necessary for the reduction oramelioration of the advancement or progression of a given disease,reduction or amelioration of the recurrence, development or onset of agiven disease, and/or to improve or enhance the prophylactic ortherapeutic effect(s) of another therapy (e.g., a therapy other thananti-hOX40L antibody provided herein). In some embodiments, theeffective amount of an antibody of the invention is from about 0.1 mg/kg(mg of antibody per kg weight of the subject) to about 100 mg/kg. Incertain embodiments, an effective amount of an antibody provided thereinis about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg,about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg,about 60 mg/kg, about 70 mg/kg, about 80 mg/kg about 90 mg/kg or about100 mg/kg (or a range therein). In some embodiments, “effective amount”as used herein also refers to the amount of an antibody of the inventionto achieve a specified result (e.g., inhibition of a hOX40L biologicalactivity of a cell, such as inhibition of secretion of CCL20, IL-8 orRANTES, or INF-γ, TNF-α or IL-2, in particular INF-γ from the cell).

The term “epitope” as used herein refers to a localized region on thesurface of an antigen, such as hOX40L polypeptide or hOX40L polypeptidefragment, that is capable of being bound to one or more antigen bindingregions of an antibody, and that has antigenic or immunogenic activityin an animal, preferably a mammal, and most preferably in a human, thatis capable of eliciting an immune response. An epitope havingimmunogenic activity is a portion of a polypeptide that elicits anantibody response in an animal. An epitope having antigenic activity isa portion of a polypeptide to which an antibody specifically binds asdetermined by any method well known in the art, for example, by theimmunoassays described herein. Antigenic epitopes need not necessarilybe immunogenic. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains and havespecific three dimensional structural characteristics as well asspecific charge characteristics. A region of a polypeptide contributingto an epitope may be contiguous amino acids of the polypeptide or theepitope may come together from two or more non-contiguous regions of thepolypeptide. The epitope may or may not be a three-dimensional surfacefeature of the antigen. In certain embodiments, a hOX40L epitope is athree-dimensional surface feature of a hOX40L polypeptide (e.g., in atrimeric form of a hOX40L polypeptide). In other embodiments, a hOX40Lepitope is linear feature of a hOX40L polypeptide (e.g., in a trimericform or monomeric form of the hOX40L polypeptide). Antibodies providedherein may specifically bind to an epitope of the monomeric (denatured)form of hOX40L, an epitope of the trimeric (native) form of hOX40L, orboth the monomeric (denatured) form and the trimeric (native) form ofhOX40L. In specific embodiments, the antibodies provided hereinspecifically bind to an epitope of the trimeric form of hOX40L but donot specifically bind the monomeric form of hOX40L.

The term “excipients” as used herein refers to inert substances whichare commonly used as a diluent, vehicle, preservatives, binders, orstabilizing agent for drugs and includes, but not limited to, proteins(e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamicacid, lysine, arginine, glycine, histidine, etc.), fatty acids andphospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants(e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g.,sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol,sorbitol, etc.). See, also, Remington's Pharmaceutical Sciences (1990)Mack Publishing Co., Easton, Pa., which is hereby incorporated byreference in its entirety.

In the context of a peptide or polypeptide, the term “fragment” as usedherein refers to a peptide or polypeptide that comprises less than thefull length amino acid sequence. Such a fragment may arise, for example,from a truncation at the amino terminus, a truncation at the carboxyterminus, and/or an internal deletion of a residue(s) from the aminoacid sequence. Fragments may, for example, result from alternative RNAsplicing or from in vivo protease activity. In certain embodiments,hOX40L fragments include polypeptides comprising an amino acid sequenceof at least 5 contiguous amino acid residues, at least 10 contiguousamino acid residues, at least 15 contiguous amino acid residues, atleast 20 contiguous amino acid residues, at least 25 contiguous aminoacid residues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least 80 contiguous aminoacid residues, at least 90 contiguous amino acid residues, at leastcontiguous 100 amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, or at least 250 contiguous amino acid residues of the aminoacid sequence of a hOX40L polypeptide or an antibody that specificallybinds to a hOX40L polypeptide. In a specific embodiment, a fragment of ahOX40L polypeptide or an antibody that specifically binds to a hOX40Lantigen retains at least 1, at least 2, or at least 3 functions of thepolypeptide or antibody.

The terms “fully human antibody” or “human antibody” are usedinterchangeably herein and refer to an antibody that comprises a humanvariable region and, most preferably a human constant region. Inspecific embodiments, the terms refer to an antibody that comprises avariable region and constant region of human origin. “Fully human”anti-hOX40L antibodies, in certain embodiments, can also encompassantibodies which bind hOX40L polypeptides and are encoded by nucleicacid sequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence. In a specific embodiment,the anti-hOX40L antibodies provided herein are fully human antibodies.The term “fully human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).Exemplary methods of producing fully human antibodies are provided,e.g., in the Examples herein, but any method known in the art may beused.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse or cow) that is transgenic and/or transchromosomal forhuman immunoglobulin genes (see e.g., Taylor, L. D. et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies can have variable and constant regions derived fromhuman germline immunoglobulin sequences (See Kabat, E. A. et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

The term “fusion protein” as used herein refers to a polypeptide thatcomprises an amino acid sequence of an antibody and an amino acidsequence of a heterologous polypeptide or protein (i.e., a polypeptideor protein not normally a part of the antibody (e.g., a non-anti-hOX40Lantigen antibody)). The term “fusion” when used in relation to hOX40L orto an anti-hOX40L antibody refers to the joining of a peptide orpolypeptide, or fragment, variant and/or derivative thereof, with aheterologous peptide or polypeptide. Preferably, the fusion proteinretains the biological activity of the hOX40L or anti-hOX40L antibody.In certain embodiments, the fusion protein comprises a hOX40L antibodyVH domain, VL domain, VH CDR (one, two or three VH CDRs), and/or VL CDR(one, two or three VL CDRs), wherein the fusion protein specificallybinds to a hOX40L epitope.

The term “heavy chain” when used in reference to an antibody refers tofive distinct types, called alpha (α), delta (δ), epsilon (ε), gamma (γ)and mu (μ), based on the amino acid sequence of the heavy chain constantdomain. These distinct types of heavy chains are well known and giverise to five classes of antibodies, IgA, IgD, IgE, IgG and IgM,respectively, including four subclasses of IgG, namely IgG1, IgG1, IgG3and IgG4. Preferably the heavy chain is a human heavy chain. In oneexample, the heavy chain is a disabled IgG isotype, e.g. a disabledIgG4. In certain embodiments, the antibodies of the invention comprise ahuman gamma 4 constant region. In another embodiment, the heavy chainconstant region does not bind Fc-γ receptors, and e.g. comprises aLeu235Glu mutation. In another embodiment, the heavy chain constantregion comprises a Ser228Pro mutation to increase stability. In anotherembodiment, the heavy chain constant region is IgG4-PE.

The term “host” as used herein refers to an animal, preferably a mammal,and most preferably a human.

The term “host cell” as used herein refers to the particular subjectcell transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell. Progeny of such a cell may not beidentical to the parent cell transfected with the nucleic acid moleculedue to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid molecule intothe host cell genome.

The term “immunomodulatory agent” and variations thereof including, butnot limited to, immunomodulatory agents, as used herein refer to anagent that modulates a host's immune system. In certain embodiments, animmunomodulatory agent is an immunosuppressant agent. In certain otherembodiments, an immunomodulatory agent is an immunostimulatory agent. Inaccordance with the invention, an immunomodulatory agent used in thecombination therapies of the invention does not include an anti-hOX40Lantibody or antigen-binding fragment. Immunomodulatory agents include,but are not limited to, small molecules, peptides, polypeptides,proteins, fusion proteins, antibodies, inorganic molecules, mimeticagents, and organic molecules.

As used herein, the term “in combination” in the context of theadministration of other therapies refers to the use of more than onetherapy. The use of the term “in combination” does not restrict theorder in which therapies are administered to a subject with a disease. Afirst therapy can be administered before (e.g., 1 minute, 45 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks), concurrently, or after (e.g., 1minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) theadministration of a second therapy to a subject which had, has, or issusceptible to a hOX40L-mediated disease. Any additional therapy can beadministered in any order with the other additional therapies. Incertain embodiments, the antibodies of the invention can be administeredin combination with one or more therapies (e.g., therapies that are notthe antibodies of the invention that are currently administered toprevent, treat, manage, and/or ameliorate a hOX40L-mediated disease.Non-limiting examples of therapies that can be administered incombination with an antibody of the invention include analgesic agents,anesthetic agents, antibiotics, or immunomodulatory agents or any otheragent listed in the U.S. Pharmacopoeia and/or Physician's DeskReference.

An “isolated” or “purified” antibody is for example substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the antibody is derived, or substantially freeof chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of an antibody in which the antibody is separated fromcellular components of the cells from which it is isolated orrecombinantly produced. Thus, an antibody that is substantially free ofcellular material includes preparations of antibody having less thanabout 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (alsoreferred to herein as a “contaminating protein”). When the antibody isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the antibodyis produced by chemical synthesis, it is preferably substantially freeof chemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the antibodyhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the antibody of interest. In apreferred embodiment, antibodies of the invention are isolated orpurified.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. In a specific embodiment, a nucleic acidmolecule(s) encoding an antibody of the invention is isolated orpurified.

The term “human OX40L,” “hOX40L” or “hOX40L polypeptide” and similarterms refers to the polypeptides (“polypeptides,” “peptides” and“proteins” are used interchangeably herein) comprising the amino acidsequence in the sequence listing and related polypeptides, including SNPvariants thereof. Related polypeptides include allelic variants (e.g.,SNP variants); splice variants; fragments; derivatives; substitution,deletion, and insertion variants; fusion polypeptides; and interspecieshomologs, preferably, which retain hOX40L activity and/or are sufficientto generate an anti-hOX40L immune response. Also encompassed are solubleforms of hOX40L which are sufficient to generate an anti-hOX40Limmunological response. As those skilled in the art will appreciate, ananti-hOX40L antibody of the invention can bind to a hOX40L polypeptide,polypeptide fragment, antigen, and/or epitope, as an epitope is part ofthe larger antigen, which is part of the larger polypeptide fragment,which, in turn, is part of the larger polypeptide hOX40L can exist in atrimeric (native) or monomeric (denatured) form.

The terms “Kabat numbering,” and like terms are recognized in the artand refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy chain variable regions of an antibody, or an antigen bindingportion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and,Kabat et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242). For the heavy chain variable region, thehypervariable region typically ranges from amino acid positions 31 to 35for CDR1, amino acid positions 50 to 65 for CDR2, and amino acidpositions 95 to 102 for CDR3.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of homogenous or substantially homogeneous antibodies, andeach monoclonal antibody will typically recognize a single epitope onthe antigen. In preferred embodiments, a “monoclonal antibody,” as usedherein, is an antibody produced by a single hybridoma or other cell,wherein the antibody specifically binds to only a hOX40L epitope asdetermined, e.g., by ELISA or other antigen-binding or competitivebinding assay known in the art or in the Examples provided herein. Theterm “monoclonal” is not limited to any particular method for making theantibody. For example, monoclonal antibodies of the invention may bemade by the hybridoma method as described in Kohler et al.; Nature,256:495 (1975) or may be isolated from phage libraries using thetechniques as described herein, for example. Other methods for thepreparation of clonal cell lines and of monoclonal antibodies expressedthereby are well known in the art (see, for example, Chapter 11 in:Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al.,eds., John Wiley and Sons, New York). Other exemplary methods ofproducing other monoclonal antibodies are provided in the Examplesherein.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to those which are found in nature and notmanipulated by a human being.

The term “pharmaceutically acceptable” as used herein means beingapproved by a regulatory agency of the Federal or a state government, orlisted in the U.S. Pharmacopeia, European Pharmacopeia or othergenerally recognized Pharmacopeia for use in animals, and moreparticularly in humans.

“Polyclonal antibodies” as used herein refers to an antibody populationgenerated in an immunogenic response to a protein having many epitopesand thus includes a variety of different antibodies directed to the sameand to different epitopes within the protein. Methods for producingpolyclonal antibodies are known in the art (See, e.g., see, for example,Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed.,Ausubel et al., eds., John Wiley and Sons, New York).

As used herein, the term “polynucleotide,” “nucleotide,” nucleic acid”“nucleic acid molecule” and other similar terms are used interchangeableand include DNA, RNA, mRNA and the like.

As used herein, the terms “prevent,” “preventing,” and “prevention”refer to the total or partial inhibition of the development, recurrence,onset or spread of a hOX40L-mediated disease and/or symptom relatedthereto, resulting from the administration of a therapy or combinationof therapies provided herein (e.g., a combination of prophylactic ortherapeutic agents, such as an antibody of the invention).

As used herein, the term “prophylactic agent” refers to any agent thatcan totally or partially inhibit the development, recurrence, onset orspread of a hOX40L-mediated disease and/or symptom related thereto in asubject. In certain embodiments, the term “prophylactic agent” refers toan antibody of the invention. In certain other embodiments, the term“prophylactic agent” refers to an agent other than an antibody of theinvention. Preferably, a prophylactic agent is an agent which is knownto be useful to or has been or is currently being used to prevent ahOX40L-mediated disease and/or a symptom related thereto or impede theonset, development, progression and/or severity of a hOX40L-mediateddisease and/or a symptom related thereto. In specific embodiments, theprophylactic agent is a fully human anti-hOX40L antibody, such as afully human anti-hOX40L monoclonal antibody.

In an embodiment, the prophylaxis prevents the onset of the disease orcondition or of the symptoms of the disease or condition. In oneembodiment, the prophylactic treatment prevents the worsening, or onset,of the disease or condition. In one embodiment, the prophylactictreatment prevents the worsening of the disease or condition.

In another embodiment, an anti-OX40L antibody of the invention isadministered intravenously (e.g. before or concomitantly with atransplant, e.g. blood or organ transplant). In another embodiment, saidantibody is administered at a dose of about 5-10 mg/kg (e.g. at about 8mg/kg). In another embodiment, said antibody is administered at a doseselected from about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg,5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg,about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70mg/kg, about 80 mg/kg about 90 mg/kg or about 100 mg/kg, in particularabout 1 mg/kg, or about 3 mg/kg.

In another embodiment, said antibody is administered 1-4 days beforetransplant (e.g. of blood or organs), e.g. 1-3 days before transplant or1-2 days before transplant. In another embodiment, said antibody isadministered weekly, bi-weekly or monthly following transplant, e.g.bi-weekly. In a further embodiment, said antibody is administeredintravenously prophylactically 1-3 days before transplant at a dose ofabout 5-10 mg/kg (e.g. about 8 mg/kg) and then intravenously, bi-weeklyat a dose of about 5-10 mg/kg (e.g. about 8 mg/kg).

In another embodiment, the patient is monitored periodicallypost-transplant, for the presence of a biomarker predictive for thedevelopment of transplant rejection or of GvHD (e.g. acute GvHD), andthe anti-OX40L antibody of the invention is administered once thebiomarker levels are such that the patient is determined to be at riskof developing transplant rejection or of GvHD (e.g. acute GvHD). Thisstrategy would avoid unnecessary dosing of drug and unnecessarysuppression of the immune system. Examples of biomarkers which may beuseful as predictive biomarkers of acute GvHD may be those identified inLevine et al., “A prognostic score for acute graft-versus-host diseasebased on biomarkers: a multicentre study”, Lancet Haematol 2015;2:e21-29. These biomarkers include, but are not limited to TNFR1, ST-2,elafin and IL2Rα and Reg3α.

A region of a hOX40L contributing to an epitope may be contiguous aminoacids of the polypeptide or the epitope may come together from two ormore non-contiguous regions of the polypeptide. The epitope may or maynot be a three-dimensional surface feature of the antigen. A localizedregion on the surface of a hOX40L antigen that is capable of elicitingan immune response is a hOX40L epitope. The epitope may or may not be athree-dimensional surface feature of the antigen.

A “hOX40L-mediated disease” and “hOX40L-mediated condition” are usedinterchangeably and refer to any disease or condition that is completelyor partially caused by or is the result of hOX40L. In certainembodiments, hOX40L is aberrantly (e.g., highly) expressed on thesurface of a cell. In some embodiments, hOX40L may be aberrantlyupregulated on a particular cell type. In other embodiments, normal,aberrant or excessive cell signaling is caused by binding of hOX40L to ahOX40L ligand. In certain embodiments, the hOX40L ligand is OX40, forexample, that is expressed on the surface of a cell, such as a colonicepithelial cell. In certain embodiments, the hOX40L-mediated disease isan inflammatory bowel disease (IBD), such as Crohn's disease (CD) orulcerative colitis (UC). In other embodiments, the hOX40L-mediateddisease is graft-versus-host disease (GVHD). In other embodiments, thehOX40L-mediated disease is selected from pyoderma gangrenosum, giantcell arteritis, Schnitzler syndrome, non-infectious scleritis anduveitis (non-infectious/autoimmune and/or systemic). In otherembodiments, a hOX40L mediated disease or condition selected from anautoimmune disease or condition, a systemic inflammatory disease orcondition, or transplant rejection; for example inflammatory boweldisease (IBD), Crohn's disease, rheumatoid arthritis, transplantrejection, allogeneic transplant rejection, graft-versus-host disease(GvHD), ulcerative colitis, systemic lupus erythematosus (SLE),diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity,multiple sclerosis and atherosclerosis, in particular GvHD.

The terms “hOX40L receptor” or “hOX40L binding receptor” are usedinterchangeably herein and refer to a receptor polypeptide that binds tohOX40L. In specific embodiments, the hOX40L receptor is Hox40. In someembodiments, the hOX40L receptor is expressed on the surface of a cell,such as a colonic epithelial cell; or on graft or transplant tissue oron host tissue.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or aprimate (e.g., monkey and human), most preferably a human. In oneembodiment, the subject is a mammal, preferably a human, having ahOX40L-mediated disease. In another embodiment, the subject is a mammal,preferably a human, at risk of developing a hOX40L-mediated disease.

As used herein “substantially all” refers to refers to at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or about 100%.

The term “substantially free of surfactant” as used herein refers to aformulation of an antibody that specifically binds to a hOX40L antigen,said formulation containing less than 0.0005%, less than 0.0003%, orless than 0.0001% of surfactants and/or less than 0.0005%, less than0.0003%, or less than 0.0001% of surfactants.

The term “substantially free of salt” as used herein refers to aformulation of an antibody that specifically binds to a hOX40L antigen,said formulation containing less than 0.0005%, less than 0.0003%, orless than 0.0001% of inorganic salts.

The term “surfactant” as used herein refers to organic substances havingamphipathic structures; namely, they are composed of groups of opposingsolubility tendencies, typically an oil-soluble hydrocarbon chain and awater-soluble ionic group. Surfactants can be classified, depending onthe charge of the surface-active moiety, into anionic, cationic, andnonionic surfactants. Surfactants are often used as wetting,emulsifying, solubilizing, and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.

As used herein, the term “tag” refers to any type of moiety that isattached to, e.g., a polypeptide and/or a polynucleotide that encodes ahOX40L or hOX40L antibody or antigen binding fragment thereof. Forexample, a polynucleotide that encodes a hOX40L, hOX40L antibody orantigen binding fragment thereof can contain one or more additionaltag-encoding nucleotide sequences that encode a, e.g., a detectablemoiety or a moiety that aids in affinity purification. When translated,the tag and the antibody can be in the form of a fusion protein. Theterm “detectable” or “detection” with reference to a tag refers to anytag that is capable of being visualized or wherein the presence of thetag is otherwise able to be determined and/or measured (e.g., byquantitation). A non-limiting example of a detectable tag is afluorescent tag.

As used herein, the term “therapeutic agent” refers to any agent thatcan be used in the treatment, management or amelioration of ahOX40L-mediated disease and/or a symptom related thereto. In certainembodiments, the term “therapeutic agent” refers to an antibody of theinvention. In certain other embodiments, the term “therapeutic agent”refers to an agent other than an antibody of the invention. Preferably,a therapeutic agent is an agent which is known to be useful for, or hasbeen or is currently being used for the treatment, management oramelioration of a hOX40L-mediated disease or one or more symptomsrelated thereto. In specific embodiments, the therapeutic agent is afully human anti-hOX40L antibody, such as a fully human anti-hOX40Lmonoclonal antibody.

The combination of therapies (e.g., use of prophylactic or therapeuticagents) which is more effective than the additive effects of any two ormore single therapy. For example, a synergistic effect of a combinationof prophylactic and/or therapeutic agents permits the use of lowerdosages of one or more of the agents and/or less frequent administrationof said agents to a subject with a hOX40L-mediated disease. The abilityto utilize lower dosages of prophylactic or therapeutic therapies and/orto administer said therapies less frequently reduces the toxicityassociated with the administration of said therapies to a subjectwithout reducing the efficacy of said therapies in the prevention,management, treatment or amelioration of a hOX40L-mediated disease. Inaddition, a synergistic effect can result in improved efficacy oftherapies in the prevention, or in the management, treatment oramelioration of a hOX40L-mediated disease. Finally, synergistic effectof a combination of therapies (e.g., prophylactic or therapeutic agents)may avoid or reduce adverse or unwanted side effects associated with theuse of any single therapy.

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and a further therapeutic agents independently selectedfrom the group consisting of rapamycin (sirolimus), tacrolimus,ciclosporin, corticosteroids (e.g. methylprednisolone), methotrexate,mycophenolate mofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies(e.g. ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30antibodies (e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5receptor antagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat. In another embodimentthe combination comprises an anti-OX40L antibody of the invention and afurther therapeutic agents independently selected from the groupconsisting of rapamycin (sirolimus), tacrolimus, ciclosporin,corticosteroids (e.g. methylprednisolone), methotrexate, mycophenolatemofetil, anti-CD28 antibodies, CTLA4-Fc molecules (e.g. abatacept),anti-CD40L antibodies, anti-LFA1 antibodies, anti-CD52 antibodies (e.g.alemtuzumab), cyclophosphamide and anti-thymocyte globulins.

In some embodiments the combination comprises an anti-OX40L antibody ofthe invention and further therapeutic agents independently selected fromthe group consisting of calcineurin inhibitors (e.g. tacrolimus,ciclosporin), mTOR inhibitors (e.g. rapamycin (sirolimus)), andantiproliferative agents (e.g. mycophenolate mofetil, cyclophosphamide).

In further embodiments the combination comprises an anti-OX40L antibodyof the invention and further therapeutic agents independently selectedfrom the group consisting of immunosuppressants that modulate IL-2signalling (e.g. tacrolimus, ciclosporin, rapamycin (sirolimus), andanti-CD25 antibodies (e.g. basilixumab, daclizumab).

Without being bound by theory, it is thought that the mechanism ofaction of an anti-OX40L antibody of the invention is complementary tofurther therapeutic agents which modulate immune function. Inparticular, agents that modulate IL-2 signalling or that inhibitIL-2/IL-2R-mediated T cell proliferation may synergistically combinewith an anti-OX40L antibody resulting in greater immune modulation thanwould be observed with either agent alone. As shown in Examples 7 and 9hereinbelow, both tacrolimus and rapamycin display immune modulatingactivity. Tacrolimus and rapamycin are both agents which are known tomodulate IL-2 signalling. In particular, rapamycin is known to act as anmTOR inhibitor, which reduces IL-2 and IL2R transcription, and inhibitscell cycle progression evoked by IL2R activation, but there may be othermechanisms on proliferation of T cells by which mTOR inhibitors mayfunction (Thomson et al, Nat. Rev. Immunol., 2009, 9(5), 324-337;Scheffert & Raza, J. Thorac. Dis., 2014, 6(8), 1039-1053). FIG. 6 hereinshows that the mechanism of an anti-OX40L antibody is different withregards to Tscm population to both these agents, and FIG. 7 shows asynergistic effect on survival of an anti-OX40L antibody of theinvention in combination with rapamycin. It is therefore thought thatother agents having a similar mechanism of action to rapamycin and/ortacrolimus will also result in a synergistic effect when used incombination with the anti-OX40L antibodies of the invention.

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and rapamycin (sirolimus).

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and tacrolimus. In one embodiment, the combinationcomprises an anti-OX40L antibody of the invention and a combination oftacrolimus and methotrexate. In another embodiment, the combinationcomprises an anti-OX40L antibody of the invention and ciclosporin. Inanother embodiment, the combination comprises an anti-OX40L antibody ofthe invention and ciclosporin and methotrexate. In another embodiment,the combination comprises an anti-OX40L antibody of the invention andcyclophosphamide. In another embodiment, the combination comprises ananti-OX40L antibody of the invention and mycophenolate mofetil.

As used herein, the term “therapy” refers to any protocol, method and/oragent that can be used in the prevention, management, treatment and/oramelioration of a hOX40L-mediated disease (e.g., IBD or GVHD). Incertain embodiments, the terms “therapies” and “therapy” refer to abiological therapy, supportive therapy, and/or other therapies useful inthe prevention, management, treatment and/or amelioration of ahOX40L-mediated disease known to one of skill in the art such as medicalpersonnel.

As used herein, the terms “treat,” “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity, and/orduration of a hOX40L-mediated disease (e.g., IBD or GVHD) resulting fromthe administration of one or more therapies (including, but not limitedto, the administration of one or more prophylactic or therapeuticagents, such as an antibody of the invention). In specific embodiments,such terms refer to the reduction or inhibition of the binding of hOX40Lto OX40, the reduction or inhibition of the production or secretion ofCCL20 from a cell expressing hOX40 or hOX40L, the reduction orinhibition of the production or secretion of IL-8 from a cell expressinghOX40 or hOX40L, the reduction or inhibition of the production orsecretion of RANTES from a cell expressing hOX40 or hOX40L, and/or theinhibition or reduction of one or more symptoms associated with ahOX40L-mediated disease, such as an IBD or GVHD. In specificembodiments, such terms refer to the reduction or inhibition of thebinding of hOX40L to OX40, the reduction or inhibition of the productionor secretion of INF-γ from a cell expressing hOX40 or hOX40L, thereduction or inhibition of the production or secretion of TNF-α from acell expressing hOX40 or hOX40L, the reduction or inhibition of theproduction or secretion of IL-2 from a cell expressing hOX40 or hOX40L,and/or the inhibition or reduction of one or more symptoms associatedwith a hOX40L-mediated disease, such as an IBD or GVHD (in particularGvHD). In an example, the cell is a human cell. In specific embodiments,a prophylactic agent is a fully human anti-hOX40L antibody, such as afully human anti-hOX40L monoclonal antibody.

The term “variable region” or “variable domain” refers to a portion ofthe OX40L and heavy chains, typically about the amino-terminal 120 to130 amino acids in the heavy chain and about 100 to 110 amino acids inthe light chain, which differ extensively in sequence among antibodiesand are used in the binding and specificity of each particular antibodyfor its particular antigen. The variability in sequence is concentratedin those regions called complimentarily determining regions (CDRs) whilethe more highly conserved regions in the variable domain are calledframework regions (FR). The CDRs of the OX40L and heavy chains areprimarily responsible for the interaction of the antibody with antigen.Numbering of amino acid positions used herein is according to the EUIndex, as in Kabat et al. (1991) Sequences of proteins of immunologicalinterest. (U.S. Department of Health and Human Services, Washington,D.C.) 5th ed. (“Kabat et al.”). In preferred embodiments, the variableregion is a human variable region.

Antibodies

Antibodies of the invention include, but are not limited to, syntheticantibodies, monoclonal antibodies, recombinantly produced antibodies,multispecific antibodies (including bi-specific antibodies), humanantibodies, humanized antibodies, chimeric antibodies, intrabodies,single-chain Fvs (scFv) (e.g., including monospecific, bispecific,etc.), camelized antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above.

In particular, antibodies provided herein include immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatspecifically binds to a hOX40L antigen. The immunoglobulin moleculesprovided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA andIgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. In a specific embodiment, an antibody providedherein is an IgG antibody, preferably an IgG1 or IgG4. In certainembodiments, the antibodies of the invention comprise a human gamma 4constant region. In another embodiment, the heavy chain constant regiondoes not bind Fc-γ receptors, and e.g. comprises a Leu235Glu mutation.In another embodiment, the heavy chain constant region comprises aSer228Pro mutation to increase stability. In another embodiment, theheavy chain constant region is IgG4-PE.

Variants and derivatives of antibodies include antibody fragments thatretain the ability to specifically bind to an epitope. Preferredfragments include Fab fragments; Fab′ (an antibody fragment containing asingle anti-binding domain comprising an Fab and an additional portionof the heavy chain through the hinge region); F(ab′)₂ (two Fab′molecules joined by interchain disulfide bonds in the hinge regions ofthe heavy chains; the Fab′ molecules may be directed toward the same ordifferent epitopes); a bispecific Fab (a Fab molecule having two antigenbinding domains, each of which may be directed to a different epitope);a single chain Fab chain comprising a variable region, also known as, asFv; a disulfide-linked Fv, or dsFv; a camelized VH (the variable,antigen-binding determinative region of a single heavy chain of anantibody in which some amino acids at the VH interface are those foundin the heavy chain of naturally occurring camel antibodies); abispecific sFv (a sFv or a dsFv molecule having two antigen-bindingdomains, each of which may be directed to a different epitope); adiabody (a dimerized sFv formed when the VH domain of a first sFvassembles with the VL domain of a second sFv and the VL domain of thefirst sFv assembles with the VH domain of the second sFv; the twoantigen-binding regions of the diabody may be directed towards the sameor different epitopes); and a triabody (a trimerized sFv, formed in amanner similar to a diabody, but in which three antigen-binding domainsare created in a single complex; the three antigen binding domains maybe directed towards the same or different epitopes). Derivatives ofantibodies also include one or more CDR sequences of an antibodycombining site. The CDR sequences may be linked together on a scaffoldwhen two or more CDR sequences are present. In certain embodiments, theantibody to be used with the invention comprises a single-chain Fv(“scFv”). scFvs are antibody fragments comprising the VH and VL domainsof an antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvssee Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The antibodies of the invention may be from any animal origin includingbirds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken). In certain embodiments, theantibodies of the invention are human or humanized monoclonalantibodies. As used herein, “human” antibodies include antibodies havingthe amino acid sequence of a human immunoglobulin and include antibodiesisolated from human immunoglobulin libraries or from mice that expressantibodies from human genes.

In preferred embodiments, the antibodies of the invention are fullyhuman antibodies, such as fully human antibodies that specifically binda hOX40L polypeptide, a hOX40L polypeptide fragment, or a hOX40Lepitope. Such fully human antibodies would be advantageous over fullymouse (or other full or partial non-human species antibodies), humanizedantibodies, or chimeric antibodies to minimize the development ofunwanted or unneeded side effects, such as immune responses directedtoward non-fully human antibodies (e.g., anti-hOX40L antibodies derivedfrom other species) when administered to the subject.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a hOX40L polypeptide or may bespecific for both a hOX40L polypeptide as well as for a heterologousepitope, such as a heterologous polypeptide or solid support material.In preferred embodiments, the antibodies provided herein aremonospecific for a given epitope of a hOX40L polypeptide and do notspecifically bind to other epitopes.

Also provided herein is a B-cell (e.g., an immortalised B-cell) or ahybridoma that produces an anti-hOX40L antibody or fragment describedherein.

In certain embodiments, an isolated antibody is provided herein thatspecifically binds to a hOX40L epitope wherein the binding to the hOX40Lepitope by the antibody is competitively blocked (e.g., in adose-dependent manner) by an antibody or fragment of the invention. Theantibody may or may not be a fully human antibody. In preferredembodiments, the antibody is a fully human monoclonal anti-hOX40Lantibody, and even more preferably a fully human, monoclonal, antagonistanti-hOX40L antibody. Exemplary competitive blocking tests that can beused are provided in the Examples herein.

In some embodiments, the antibody or fragment of the invention competes(e.g., in a dose-dependent manner) with OX40 Receptor (or a fusionprotein thereof) for binding to cell surface-expressed hOX40L. In otherembodiments, the antibody or fragment of the invention competes (e.g.,in a dose-dependent manner) with OX40 Receptor (or a fusion proteinthereof) for binding to soluble hOX40L. Exemplary competitive bindingassays that can be used are provided in the Examples herein. In oneembodiment, the antibody or fragment partially or completely inhibitsbinding of hOX40 to cell surface-expressed OX40L, such as hOX40L. Inanother embodiment, the antibody partially or completely inhibitsbinding of hOX40 to soluble hOX40L. In some embodiments, the antibody orfragment partially or completely inhibits the secretion of CCL20, IL-8,and/or RANTES, or INF-γ, TNF-α or IL-2, in particular INF-γ from a cellhaving cell surface-expressed OX40. In certain embodiments, the cellexpressing the OX40 is a colonic epithelial cell.

Preferably, the antibodies of the invention are fully human, monoclonalantibodies, such as fully human, monoclonal antagonist antibodies, thatspecifically bind to hOX40L.

In some embodiments, the antibody or fragment provided herein binds to ahOX40L epitope that is a three-dimensional surface feature of a hOX40Lpolypeptide (e.g., in a trimeric form of a hOX40L polypeptide). A regionof a hOX40L polypeptide contributing to an epitope may be contiguousamino acids of the polypeptide or the epitope may come together from twoor more non-contiguous regions of the polypeptide A hOX40L epitope maybe present in (a) the trimeric form (“a trimeric hOX40L epitope”) ofhOX40L, (b) the monomeric form (“a monomeric hOX40L epitope”) of hOX40L,(c) both the trimeric and monomeric form of hOX40L, (d) the trimericform, but not the monomeric form of hOX40L, or (e) the monomeric form,but not the trimeric form of hOX40L.

For example, in some embodiments, the epitope is only present oravailable for binding in the trimeric (native) form, but is not presentor available for binding in the monomeric (denatured) form by ananti-hOX40L antibody. In other embodiments, the hOX40L epitope is linearfeature of the hOX40L polypeptide (e.g., in a trimeric form or monomericform of the hOX40L polypeptide). Antibodies provided herein mayspecifically bind to (a) an epitope of the monomeric form of hOX40L, (b)an epitope of the trimeric form of hOX40L, (c) an epitope of themonomeric but not the trimeric form of hOX40L, (d) an epitope of thetrimeric but not the monomeric form of hOX40L, or (e) both the monomericform and the trimeric form of hOX40L. In preferred embodiments, theantibodies provided herein specifically bind to an epitope of thetrimeric form of hOX40L but do not specifically bind to an epitope themonomeric form of hOX40L.

The present invention also provides antibodies that specifically bind toa hOX40L epitope, the antibodies comprising derivatives of the VHdomains, VH CDRs, VL domains, and VL CDRs described herein thatspecifically bind to a hOX40L antigen. The present invention alsoprovides antibodies comprising derivatives of antibodies disclosed inthe Examples, wherein said antibodies specifically bind to a hOX40Lepitope. Standard techniques known to those of skill in the art can beused to introduce mutations in the nucleotide sequence encoding amolecule of the invention, including, for example, site-directedmutagenesis and PCR-mediated mutagenesis which results in amino acidsubstitutions. Preferably, the derivatives include less than 25 aminoacid substitutions, less than 20 amino acid substitutions, less than 15amino acid substitutions, less than 10 amino acid substitutions, lessthan 5 amino acid substitutions, less than 4 amino acid substitutions,less than 3 amino acid substitutions, or less than 2 amino acidsubstitutions relative to the original molecule. In another embodiment,the derivatives have conservative amino acid substitutions. In apreferred embodiment, the derivatives have conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. Alternatively, mutations can be introduced randomly along allor part of the coding sequence, such as by saturation mutagenesis, andthe resultant mutants can be screened for biological activity toidentify mutants that retain activity. Following mutagenesis, theencoded protein can be expressed and the activity of the protein can bedetermined.

In another embodiment, an antibody that specifically binds to a hOX40Lepitope comprises a variable domain amino acid sequence that is at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% identical to a variabledomain amino acid sequence of the sequence listing.

In specific embodiments, the antibody is a fully human anti-humanantibody, such as a fully human monoclonal antibody. Fully humanantibodies may be produced by any method known in the art. Exemplarymethods include immunization with a hOX40L antigen (any hOX40Lpolypeptide capable of eliciting an immune response, and optionallyconjugated to a carrier) of transgenic animals (e.g., mice) that arecapable of producing a repertoire of human antibodies in the absence ofendogenous immunoglobulin production; see, e.g., Jakobovits et al.,(1993) Proc. Natl. Acad. Sci., 90:2551; Jakobovits et al., (1993)Nature, 362:255 258 (1993); Bruggermann et al., (1993) Year in Immunol.,7:33. Other methods of producing fully human anti-hOX40L antibodies canbe found in the Examples provided herein.

Alternatively, fully human antibodies may be generated through the invitro screening of phage display antibody libraries; see e.g.,Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581 (1991), incorporated herein by reference. Variousantibody-containing phage display libraries have been described and maybe readily prepared by one skilled in the art. Libraries may contain adiversity of human antibody sequences, such as human Fab, Fv, and scFvfragments, that may be screened against an appropriate target.

The antibodies and fragments of the invention include antibodies andfragments that are chemically modified, i.e., by the covalent attachmentof any type of molecule to the antibody. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenchemically modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formulation, metabolic synthesis of tunicamycin,etc. Additionally, the antibody may contain one or more non-classicalamino acids.

The present invention also provides antibodies that specifically bind toa hOX40L antigen which comprise a framework region known to those ofskill in the art (e.g., a human or non-human fragment). The frameworkregion may, for example, be naturally occurring or consensus frameworkregions. Most preferably, the framework region of an antibody of theinvention is human (see, e.g., Chothia et al., 1998, J. Mol. Biol.278:457-479 for a listing of human framework regions, which isincorporated by reference herein in its entirety). See also Kabat et al.(1991) Sequences of Proteins of Immunological Interest (U.S. Departmentof Health and Human Services, Washington, D.C.) 5th ed.

In a specific embodiment, the present invention provides for antibodiesthat specifically bind to a hOX40L antigen, said antibodies comprisingthe amino acid sequence of one or more of the CDRs in the sequencelisting (i.e. Seq ID No:4, Seq ID No:10, Seq ID No:36, Seq ID No:42, SeqID No:68, Seq ID No:74, Seq ID No:96 or Seq ID No:102, in particular,Seq ID No:36 or Seq ID No:42 for HCDR1; Seq ID No:6, Seq ID No:12, SeqID No:38, Seq ID No:44, Seq ID No:70, Seq ID No:76, Seq ID No:98 or SeqID No:104, in particular Seq ID No:38 or Seq ID No:44 for HCDR2; Seq IDNo:8, Seq ID No:14, Seq ID No:40, Seq ID No:46, Seq ID No:72, Seq IDNo:78, Seq ID No:100 or Seq ID No:106, in particular Seq ID No:40 or SeqID No:46 for HCDR3; Seq ID No:18, Seq ID No:24, Seq ID No:50, Seq IDNo:56, Seq ID No:82, Seq ID No:88, Seq ID No:110 or Seq ID No:116, inparticular Seq ID No:50 or Seq ID No:56 for LCDR1; Seq ID No:20, Seq IDNo:26, Seq ID No:52, Seq ID No:58, Seq ID No:84, Seq ID No:90, Seq IDNo:112 or Seq ID No:118, in particular Seq ID No:52 or Seq ID No:58 forLCDR2; and Seq ID No:22, Seq ID No:28, Seq ID No:54, Seq ID No:60, SeqID No:86, Seq ID No:92, Seq TD No:114 or Seq TD No: 120, in particularSeq TD No:54 or Seq TD No:60 for LCDR3) and human framework regions withone or more amino acid substitutions at one, two, three or more of thefollowing residues: (a) rare framework residues that differ between themurine antibody framework (i.e., donor antibody framework) and the humanantibody framework (i.e., acceptor antibody framework); (b) Vernier zoneresidues when differing between donor antibody framework and acceptorantibody framework; (c) interchain packing residues at the VH/VLinterface that differ between the donor antibody framework and theacceptor antibody framework; (d) canonical residues which differ betweenthe donor antibody framework and the acceptor antibody frameworksequences, particularly the framework regions crucial for the definitionof the canonical class of the murine antibody CDR loops; (e) residuesthat are adjacent to a CDR; (g) residues capable of interacting with theantigen; (h) residues capable of interacting with the CDR; and (i)contact residues between the VH domain and the VL domain. In certainembodiments, antibodies that specifically bind to a hOX40L antigencomprising the human framework regions with one or more amino acidsubstitutions at one, two, three or more of the above-identifiedresidues are antagonistic hOX40L antibodies.

The present invention encompasses antibodies that specifically bind to ahOX40L antigen, said antibodies comprising the amino acid sequence ofthe VH domain and/or VL domain in the sequence listing (i.e. Seq IDNo:2, Seq ID No:34, Seq ID No:66 or Seq ID No:94, in particular Seq IDNo:34 for VH domains; Seq ID No:16, Seq ID No:48, Seq ID No:80, or SeqID No:108, in particular Seq ID No:48 for VL domains) but havingmutations (e.g., one or more amino acid substitutions) in the frameworkregions. In certain embodiments, antibodies that specifically bind to ahOX40L antigen comprise the amino acid sequence of the VH domain and/orVL domain or an antigen-binding fragment thereof of an antibodydisclosed in the Examples with one or more amino acid residuesubstitutions in the framework regions of the VH and/or VL domains.

In some embodiments, antibodies provided herein decrease or inhibitbinding of hOX40L hOX40, and/or decrease or inhibit a hOX40L biologicalactivity, such as secretion of CCL20, IL8 and/or RANTES, or INF-γ, TNF-αor IL-2, in particular INF-γ, in subject (e.g., a human subject). Incertain embodiments, antibodies provided herein, such as a humanmonoclonal anti-hOX40L antibody, decreases or inhibits binding of asoluble or cell-surface expressed hOX40L to hOX40, and/or decreases orinhibits secretion of CCL20 and/or RANTES, or INF-γ, TNF-α or IL-2, inparticular INF-γ after contact with a soluble or cell-surface expressedhOX40L, in a subject. Blocking activity of an antibody provided hereinof hOX40L binding to hOX40 can be detected using an assay as describedin the Examples. Inhibition of biological activity of cells expressingOX40 by a hOX40L antibody provided herein can be detected using an assayas described in the Examples.

The present invention also provides for fusion proteins comprising anantibody provided herein that specifically binds to a hOX40L antigen anda heterologous polypeptide. In some embodiments, the heterologouspolypeptide to which the antibody is fused is useful for targeting theantibody to cells having cell surface-expressed hOX40L.

Antibody Conjugates and Fusion Proteins

The following discussion on conjugates and fusion proteins also appliesto fragments so that disclosure mentioning antibodies can also applymutatis mutandis to fragments of the invention.

In some embodiments, antibodies of the invention are conjugated orrecombinantly fused to a diagnostic, detectable or therapeutic agent orany other molecule. The conjugated or recombinantly fused antibodies canbe useful, e.g., for monitoring or prognosing the onset, development,progression and/or severity of a hOX40L-mediated disease as part of aclinical testing procedure, such as determining the efficacy of aparticular therapy.

Such diagnosis and detection can be accomplished, for example, bycoupling the antibody to detectable substances including, but notlimited to, various enzymes, such as, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metals using variouspositron emission tomographies, and non-radioactive paramagnetic metalions.

The present invention further encompasses uses of the antibodies of theinvention conjugated or recombinantly fused to a therapeutic moiety (orone or more therapeutic moieties). The antibody may be conjugated orrecombinantly fused to a therapeutic moiety, such as a cytotoxin, e.g.,a cytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includesany agent that is detrimental to cells. Therapeutic moieties include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine); alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP), and cisplatin);anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin); antibiotics (e.g., d actinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)); Auristatin molecules(e.g., auristatin PHE, bryostatin 1, and solastatin 10; see Woyke etal., Antimicrob. Agents Chemother. 46:3802-8 (2002), Woyke et al.,Antimicrob. Agents Chemother. 45:3580-4 (2001), Mohammad et al.,Anticancer Drugs 12:735-40 (2001), Wall et al., Biochem. Biophys. Res.Commun 266:76-80 (1999), Mohammad et al., Int. J. Oncol. 15:367-72(1999), all of which are incorporated herein by reference); hormones(e.g., glucocorticoids, progestins, androgens, and estrogens),DNA-repair enzyme inhibitors (e.g., etoposide or topotecan), kinaseinhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al.,Clin Cancer Res. 8(7):2167-76 (2002)); cytotoxic agents (e.g.,paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin andanalogues or homologs thereof and those compounds disclosed in U.S. Pat.Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196,6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769,5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745,5,728,868, 5,648,239, 5,587,459); farnesyl transferase inhibitors (e.g.,R115777, BMS-214662, and those disclosed by, for example, U.S. Pat. Nos.6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387,6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905,6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501,6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865,6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096,6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295,6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935,6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305);topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38;topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622;rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022;TAN-1518A; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506;and rebeccamycin); bulgarein; DNA minor groove binders such as Hoeschtdye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine;coralyne; beta-lapachone; BC-4-1; bisphosphonates (e.g., alendronate,cimadronte, clodronate, tiludronate, etidronate, ibandronate,neridronate, olpandronate, risedronate, piridronate, pamidronate,zolendronate) HMG-CoA reductase inhibitors, (e.g., lovastatin,simvastatin, atorvastatin, pravastatin, fluvastatin, statin,cerivastatin, lescol, lupitor, rosuvastatin and atorvastatin); antisenseoligonucleotides (e.g., those disclosed in the U.S. Pat. Nos. 6,277,832,5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenosine deaminaseinhibitors (e.g., Fludarabine phosphate and 2-Chlorodeoxyadenosine);ibritumomab tiuxetan (Zevalin®); tositumomab (Bexxar®)) andpharmaceutically acceptable salts, solvates, clathrates, and prodrugsthereof.

Further, an antibody of the invention may be conjugated or recombinantlyfused to a therapeutic moiety or drug moiety that modifies a givenbiological response. Therapeutic moieties or drug moieties are not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein, peptide, or polypeptidepossessing a desired biological activity. Such proteins may include, forexample, a toxin such as abrin, ricin A, pseudomonas exotoxin, choleratoxin, or diphtheria toxin; a protein such as tumor necrosis factor,γ-interferon, α-interferon, nerve growth factor, platelet derived growthfactor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-γ,AIM I (see, International Publication No. WO 97/33899), AIM II (see,International Publication No. WO 97/34911), Fas Ligand (Takahashi etal., 1994, J. Immunol., 6:1567-1574), and VEGF (see, InternationalPublication No. WO 99/23105), an anti-angiogenic agent, e.g.,angiostatin, endostatin or a component of the coagulation pathway (e.g.,tissue factor); or, a biological response modifier such as, for example,a lymphokine (e.g., interferon gamma, interleukin-1 interleukin-2(“IL-2”), interleukin-5 interleukin-6 (“IL-6”), interleukin-7 (“IL-7”),interleukin 9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-12(IL-12″), interleukin-15 (IL-15″), interleukin-23 (“IL-23”), granulocytemacrophage colony stimulating factor (“GM-CSF”), and granulocyte colonystimulating factor (“G-CSF”)), or a growth factor (e.g., growth hormone(“GH”)), or a coagulation agent (e.g., calcium, vitamin K, tissuefactors, such as but not limited to, Hagcman factor (factor XII),high-molecular-weight kininogen (HMWK), prekallikrein (PK), coagulationproteins-factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa,IX, IXa, X, phospholipid, and fibrin monomer).

The present invention encompasses antibodies of the inventionrecombinantly fused or chemically conjugated (covalent or non-covalentconjugations) to a heterologous protein or polypeptide (or fragmentthereof, preferably to a polypeptide of about 10, about 20, about 30,about 40, about 50, about 60, about 70, about 80, about 90 or about 100amino acids) to generate fusion proteins. In particular, the inventionprovides fusion proteins comprising an antigen-binding fragment of anantibody of the invention (e.g., a Fab fragment, Fd fragment, Fvfragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VLCDR) and a heterologous protein, polypeptide, or peptide. In oneembodiment, the heterologous protein, polypeptide, or peptide that theantibody is fused to is useful for targeting the antibody to aparticular cell type, such as a cell that expresses hOX40L or an hOX40Lreceptor. For example, an antibody that specifically binds to a cellsurface receptor expressed by a particular cell type (e.g., an immunecell) may be fused or conjugated to a modified antibody of theinvention.

A conjugated or fusion protein of the invention comprises any antibodyof the invention described herein and a heterologous polypeptide. In oneembodiment, a conjugated or fusion protein of the invention comprisesthe variable domains of an antibody disclosed in the Examples and aheterologous polypeptide.

In addition, an antibody of the invention can be conjugated totherapeutic moieties such as a radioactive metal ion, such asalpha-emitters such as ²¹³Bi or macrocyclic chelators useful forconjugating radiometal ions, including but not limited to, ¹³¹In, ¹³¹Lu,¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides. In certain embodiments, themacrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.,26(8):943-50, each incorporated by reference in their entireties.

Moreover, antibodies of the invention can be fused to marker sequences,such as a peptide to facilitate purification. In preferred embodiments,the marker amino acid sequence is a hexa-histidine peptide, such as thetag provided in a pQE vector (QIAGEN, Inc.), among others, many of whichare commercially available. As described in Gentz et al., 1989, Proc.Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine providesfor convenient purification of the fusion protein. Other peptide tagsuseful for purification include, but are not limited to, thehemagglutinin (“HA”) tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767),and the “FLAG” tag.

Methods for fusing or conjugating therapeutic moieties (includingpolypeptides) to antibodies are well known, see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies 84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol.Rev. 62:119-58; U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813;Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991;Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol.,154:5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341, 1992, which are incorporated herein by reference intheir entireties.

Fusion proteins may be generated, for example, through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention (e.g.,antibodies with higher affinities and lower dissociation rates). See,generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33;Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J.Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques24(2):308-313 (each of these patents and publications are herebyincorporated by reference in its entirety). Antibodies, or the encodedantibodies, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. A polynucleotide encoding an antibody of the inventionmay be recombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

An antibody of the invention can also be conjugated to a second antibodyto form an antibody heteroconjugate as described in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated or recombinantly fused to anantibody of the invention that specifically binds to a hOX40L antigenshould be chosen to achieve the desired prophylactic or therapeuticeffect(s). In certain embodiments, the antibody is a modified antibody.A clinician or other medical personnel should consider the followingwhen deciding on which therapeutic moiety or drug to conjugate orrecombinantly fuse to an antibody of the invention: the nature of thedisease, the severity of the disease, and the condition of the subject.

Antibodies of the invention may also be attached to solid supports,which are particularly useful for immunoassays or purification of thetarget antigen. Such solid supports include, but are not limited to,glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene.

Pharmaceutical Compositions

The following discussion on compositions also applies to fragments sothat disclosure mentioning antibodies can also apply mutatis mutandis tofragments of the invention.

Therapeutic formulations containing one or more antibodies of theinvention provided herein can be prepared for storage by mixing theantibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton,Pa.), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The antibodies of the invention provided herein can also, for example,be formulated in liposomes. Liposomes containing the molecule ofinterest are prepared by methods known in the art, such as described inEpstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al.(1980) Proc. Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045and 4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

Particularly useful immunoliposomes can be generated by the reversephase evaporation method with a lipid composition containingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of an antibody provided herein can beconjugated to the liposomes as described in Martin et al. (1982) J.Biol. Chem. 257:286-288 via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome; See Gabizon et al., (1989) J. National Cancer Inst.81(19):1484.

Formulations, such as those described herein, can also contain more thanone active compound as necessary for the particular indication beingtreated. In certain embodiments, formulations comprise an antibody ofthe invention and one or more active compounds with complementaryactivities that do not adversely affect each other. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended. For example, an antibody of the invention can becombined with one or more other therapeutic agents. Such combinedtherapy can be administered to the patient serially or simultaneously orin sequence.

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and a further therapeutic agents independently selectedfrom the group consisting of rapamycin (sirolimus), tacrolimus,ciclosporin, corticosteroids (e.g. methylprednisolone), methotrexate,mycophenolate mofetil, anti-CD28 antibodies, anti-IL12/IL-23 antibodies(e.g. ustekinumab), anti-CD20 antibodies (e.g.rituximab), anti-CD30antibodies (e.g. brentuximab), CTLA4-Fc molecules (e.g. abatacept), CCR5receptor antagonists (e.g. maraviroc), anti-CD40L antibodies, anti-VLA4antibodies (e.g. natalizumab), anti-LFA1 antibodies, fludarabine,anti-CD52 antibodies (e.g. alemtuzumab), anti-CD45 antibodies,cyclophosphamide, anti-thymocyte globulins, anti-complement C5antibodies (e.g. eculizumab), anti-a4b7 integrin antibodies (e.g.vedolizumab), anti-IL6 antibodies (e.g. tocilizumab), anti-IL2Rantibodies (e.g. basilixumab), anti-CD25 antibodies (e.g. daclizumab),anti-TNFa/TNFa-Fc molecules (e.g. etanercept, adalimumab, infliximab,golimumab or certolizumab pegol) and Vorinostat. In another embodimentthe combination comprises an anti-OX40L antibody of the invention and afurther therapeutic agents independently selected from the groupconsisting of rapamycin (sirolimus), tacrolimus, ciclosporin,corticosteroids (e.g. methylprednisolone), methotrexate, mycophenolatemofetil, anti-CD28 antibodies, CTLA4-Fc molecules (e.g. abatacept),anti-CD40L antibodies, anti-LFA1 antibodies, anti-CD52 antibodies (e.g.alemtuzumab), cyclophosphamide and anti-thymocyte globulins.

In some embodiments the combination comprises an anti-OX40L antibody ofthe invention and further therapeutic agents independently selected fromthe group consisting of calcineurin inhibitors (e.g. tacrolimus,ciclosporin), mTOR inhibitors (e.g. rapamycin (sirolimus)), andantiproliferative agents (e.g. mycophenolate mofetil, cyclophosphamide).

In further embodiments the combination comprises an anti-OX40L antibodyof the invention and further therapeutic agents independently selectedfrom the group consisting of immunosuppressants that modulate IL-2signalling (e.g. tacrolimus, ciclosporin, rapamycin (sirolimus), andanti-CD25 antibodies (e.g. basilixumab, daclizumab)

In one embodiment, the combination comprises an anti-OX40L antibody ofthe invention and rapamycin (sirolimus). In one embodiment, thecombination comprises an anti-OX40L antibody of the invention andtacrolimus. In one embodiment, the combination comprises an anti-OX40Lantibody of the invention and a combination of tacrolimus andmethotrexate. In another embodiment, the combination comprises ananti-OX40L antibody of the invention and ciclosporin. In anotherembodiment, the combination comprises an anti-OX40L antibody of theinvention and ciclosporin and methotrexate. In another embodiment, thecombination comprises an anti-OX40L antibody of the invention andcyclophosphamide. In another embodiment, the combination comprises ananti-OX40L antibody of the invention and mycophenolate mofetil.

An antibody of the invention can also be entrapped in microcapsuleprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton,Pa.

The formulations to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

Sustained-release preparations can also be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antagonist, which matrices arein the form of shaped articles, e.g., films, or microcapsule. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The pharmaceutical compositions provided herein contain therapeuticallyeffective amounts of one or more of the antibodies of the inventionprovided herein, and optionally one or more additional prophylactic oftherapeutic agents, in a pharmaceutically acceptable carrier. Suchpharmaceutical compositions are useful in the prevention, treatment,management or amelioration of a hOX40L-mediated disease, such as aninflammatory bowl disease, transplant rejection, GvHD or one or more ofthe symptoms thereof.

Pharmaceutical carriers suitable for administration of the compoundsprovided herein include any such carriers known to those skilled in theart to be suitable for the particular mode of administration.

In addition, the antibodies of the invention may be formulated as thesole pharmaceutically active ingredient in the composition or may becombined with other active ingredients (such as one or more otherprophylactic or therapeutic agents).

The compositions can contain one or more antibodies of the invention. Inone embodiment, the antibodies are formulated into suitablepharmaceutical preparations, such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers. In one embodiment, theantibodies described above are formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel (1985) Introduction to Pharmaceutical Dosage Forms, 4th Ed.,p. 126).

In the compositions, effective concentrations of one or more antibodiesor derivatives thereof is (are) mixed with a suitable pharmaceuticalcarrier. The concentrations of the compounds in the compositions areeffective for delivery of an amount, upon administration, that treats,prevents, or ameliorates a hOX40L-mediated disease or symptom thereof.

In one embodiment, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction ofcompound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

An antibody of the invention is included in the pharmaceuticallyacceptable carrier in an effective amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the patient treated. The therapeutically effective concentration canbe determined empirically by testing the compounds in in vitro and invivo systems using routine methods and then extrapolated therefrom fordosages for humans.

The concentration of antibody in the pharmaceutical composition willdepend on, e.g., the physicochemical characteristics of the antibody,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art.

In one embodiment, a therapeutically effective dosage produces a serumconcentration of antibody of from about 0.1 ng/ml to about 50-100 μg/ml.The pharmaceutical compositions, in another embodiment, provide a dosageof from about 0.001 mg to about 2000 mg of antibody per kilogram of bodyweight per day. Pharmaceutical dosage unit forms can be prepared toprovide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or2000 mg, and in one embodiment from about 10 mg to about 500 mg of theantibody and/or a combination of other optional essential ingredientsper dosage unit form.

The antibody can be administered at once, or may be divided into anumber of smaller doses to be administered at intervals of time. It isunderstood that the precise dosage and duration of treatment is afunction of the disease being treated and can be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuescan also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens can be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

Upon mixing or addition of the antibody, the resulting mixture can be asolution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The antibody is, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of the antibodysufficient to produce the desired therapeutic effect, in associationwith the required pharmaceutical carrier, vehicle or diluent. Examplesof unit-dose forms include ampoules and syringes and individuallypackaged tablets or capsules. Unit-dose forms can be administered infractions or multiples thereof. A multiple-dose form is a plurality ofidentical unit-dosage forms packaged in a single container to beadministered in segregated unit-dose form. Examples of multiple-doseforms include vials, bottles of tablets or capsules or bottles of pintsor gallons. Hence, multiple dose form is a multiple of unit-doses whichare not segregated in packaging.

In preferred embodiments, one or more anti-hOX40L antibodies of theinvention are in a liquid pharmaceutical formulation. Liquidpharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered can also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.

Dosage forms or compositions containing antibody in the range of 0.005%to 100% with the balance made up from non-toxic carrier can be prepared.Methods for preparation of these compositions are known to those skilledin the art.

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric-coated, sugar-coated or film-coated. Capsules canbe hard or soft gelatin capsules, while granules and powders can beprovided in non-effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms. Incertain embodiments, the formulations are capsules or tablets. Thetablets, pills, capsules, troches and the like can contain one or moreof the following ingredients, or compounds of a similar nature: abinder; a lubricant; a diluent; a glidant; a disintegrating agent; acolouring agent; a sweetening agent; a flavouring agent; a wettingagent; an emetic coating; and a film coating Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polyinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecrosscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Colouring agents include, for example, any ofthe approved certified water soluble FD and C dyes, mixtures thereof;and water insoluble FD and C dyes suspended on alumina hydrate.Sweetening agents include sucrose, lactose, mannitol and artificialsweetening agents such as saccharin, and any number of spray driedflavours. Flavouring agents include natural flavours extracted fromplants such as fruits and synthetic blends of compounds which produce apleasant sensation, such as, but not limited to peppermint and methylsalicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The antibodies of the invention can be provided in a composition thatprotects it/them from the acidic environment of the stomach. Forexample, the composition can be formulated in an enteric coating thatmaintains its integrity in the stomach and releases the active compoundin the intestine. The composition can also be formulated in combinationwith an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colourings and flavours.

The antibody can also be mixed with other active materials which do notimpair the desired action, or with materials that supplement the desiredaction, such as antacids, H2 blockers, and diuretics. The activeingredient is an antibody or pharmaceutically acceptable derivativethereof as described herein. Higher concentrations, up to about 98% byweight of the active ingredient may be included.

In all embodiments, tablets and capsules formulations can be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

In preferred embodiments, the formulations are liquid dosage forms.Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives.

Pharmaceutically acceptable substances used in non-effervescentgranules, to be reconstituted into a liquid oral dosage form, includediluents, sweeteners and wetting agents. Pharmaceutically acceptablesubstances used in effervescent granules, to be reconstituted into aliquid oral dosage form, include organic acids and a source of carbondioxide. Colouring and flavouring agents are used in all of the abovedosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Colouring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavouring agents include natural flavours extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is, in oneembodiment, encapsulated in a gelatin capsule. Such solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, thesolution, e.g., for example, in a polyethylene glycol, can be dilutedwith a sufficient quantity of a pharmaceutically acceptable liquidcarrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations can be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. RE28,819 and4,358,603. Briefly, such formulations include, but are not limited to,those containing a compound provided herein, a dialkylated mono- orpoly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

Parenteral administration, in one embodiment, is characterized byinjection, either subcutaneously, intramuscularly or intravenously isalso contemplated herein. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. The injectables, solutions and emulsions also contain one ormore excipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. Briefly, a compound providedherein is dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The antibody diffuses through the outer polymeric membrane in a releaserate controlling step. The amount of antibody contained in suchparenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Preparations for parenteral administration include sterile solutionsready for injection, sterile dry soluble products, such as lyophilizedpowders, ready to be combined with a solvent just prior to use,including hypodermic tablets, sterile suspensions ready for injection,sterile dry insoluble products ready to be combined with a vehicle justprior to use and sterile emulsions. The solutions may be either aqueousor nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anaesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations can be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN® 80). A sequestering or chelatingagent of metal ions includes EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles; and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations can be packaged in an ampoule, avial or a syringe with a needle. All preparations for parenteraladministration can be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, a therapeutically effective dosage is formulated to containa concentration of at least about 0.1% w/w up to about 90% w/w or more,in certain embodiments more than 1% w/w of the active compound to thetreated tissue(s).

The antibody can be suspended in micronized or other suitable form. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thecompound in the selected carrier or vehicle. The effective concentrationis sufficient for ameliorating the symptoms of the condition and may beempirically determined.

In other embodiments, the pharmaceutical formulations are lyophilizedpowders, which can be reconstituted for administration as solutions,emulsions and other mixtures. They may also be reconstituted andformulated as solids or gels.

The lyophilized powder is prepared by dissolving an antibody providedherein, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. In some embodiments, the lyophilized powder issterile. The solvent may contain an excipient which improves thestability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture can be a solution, suspension,emulsions or the like and can be formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The antibodies of the invention can be formulated as aerosols fortopical application, such as by inhalation (sec, e.g., U.S. Pat. Nos.4,044,126, 4,414,209, and 4,364,923, which describe aerosols fordelivery of a steroid useful for treatment of inflammatory diseases,particularly asthma). These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microfine powder for insufflations, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in one embodiment, have diameters ofless than 50 microns, in one embodiment less than 10 microns.

The compounds can be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

These solutions, particularly those intended for ophthalmic use, may beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts.

Other routes of administration, such as transdermal patches, includingiontophoretic and electrophoretic devices, and rectal administration,are also contemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devices,are well known to those of skill in the art. For example, such patchesare disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533,6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and5,860,957.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by moulding. The weight of a rectal suppository, inone embodiment, is about 2 to 3 gm.

Tablets and capsules for rectal administration can be manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

The antibodies and other compositions provided herein may also beformulated to be targeted to a particular tissue, receptor, or otherarea of the body of the subject to be treated. Many such targetingmethods are well known to those of skill in the art. All such targetingmethods are contemplated herein for use in the instant compositions. Fornon-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos.6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570,6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534,5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874. Insome embodiments, the anti-hOX40L antibodies of the invention aretargeted (or otherwise administered) to the colon, such as in a patienthaving or at risk of having an IBD. In some embodiments, the anti-hOX40Lantibodies of the invention are targeted (or otherwise administered) tothe eye, such as in a patient having or at risk of having uveitis.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumour-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art. For example, liposomeformulations can be prepared as described in U.S. Pat. No. 4,522,811.Briefly, liposomes such as multilamellar vesicles (MLV's) may be formedby drying down egg phosphatidyl choline and brain phosphatidyl serine(7:3 molar ratio) on the inside of a flask. A solution of a compoundprovided herein in phosphate buffered saline lacking divalent cations(PBS) is added and the flask shaken until the lipid film is dispersed.The resulting vesicles are washed to remove unencapsulated compound,pelleted by centrifugation, and then resuspended in PBS.

Methods of Administration and Dosing

The present invention further provides for compositions comprising oneor more antibodies or fragments of the invention for use in theprevention, management, treatment and/or amelioration of ahOX40L-mediated disease (or symptom thereof). Discussion in respect ofantibodies also applies mutatis mutandis to fragments of the invention.In an alternative, the present invention further provides forcompositions comprising one or more antibodies or fragments of theinvention for use in the prevention, management, treatment and/oramelioration of an OX40L-mediated disease (or symptom thereof) in asubject, wherein the OX40L is non-human (e.g., canine, feline, equine,bovine, ovine or porcine) and the subject is respectively a dog, cat,horse, cow, sheep or pig.

In certain embodiments, provided herein are compositions comprising oneor more antibodies of the invention for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated disease,such as IBD (e.g., ulcerative colitis or Crohn's disease), or a symptomthereof. IBD symptoms may range from mild to severe and generally dependupon the part of the intestinal tract involved. Exemplary symptoms ofIBD include abdominal cramps and pain, bloody diarrhoea, severe urgencyto have a bowel movement, fever, loss of appetite, weight loss, anaemia,fatigue, and/or sores on lower legs, ankles, calves, thighs, and arms.Exemplary intestinal complications of IBD include profuse bleeding fromthe ulcers, perforation or rupture of the bowel, strictures andobstruction, fistulae (abnormal passage) and perianal disease, toxicmegacolon (e.g., acute nonobstructive dilation of the colon), and/ormalignancy (e.g., cancer of the colon or small intestine). Exemplaryextraintestinal complications of IBD include arthritis, skin conditions,inflammation of the eye, liver and kidney disorders, and/or bone loss.Any combination of these symptoms may be prevented, managed, treated,and/or ameliorated using the compositions and methods provided herein.

In certain embodiments, provided herein are compositions comprising oneor more antibodies of the invention for use in the prevention,management, treatment and/or amelioration of an hOX40L-mediated disease,such as GVHD, or a symptom thereof. GVHD generally occurs followingallogeneic or matched unrelated bone marrow transplants (BMT).

In some embodiments, the GVHD is acute GVHD. The symptoms of acute GVHDcan happen quickly and can be mild or severe. In certain instances,acute GVHD develops within about three months after transplant, such aswhen blood counts recover after transplant. It certain instances, theacute GVHD affects the skin, gastrointestinal (GI) tract and/or liver.For example, in some patients, acute skin GVHD begins with a rash, forexample, on the palms of the patient's hands, soles of the feet, orshoulders. However, the rash can become widespread, and may be itchy andpainful and/or might blister and peel. Acute liver GVHD may affectnormal functions of the liver, such as liver enzymes, and may in turn,cause jaundice. Acute liver GVHD may also cause the patient's abdomen tobecome swollen and painful if the liver becomes enlarged Finally,symptoms of acute gut GVHD (or GVHD of the digestive system) can includediarrhoea, mucus or blood in the stool, cramping or abdominal pain,indigestion, nausea and/or loss of appetite. Other general symptoms ofacute GVHD can include anaemia, low grade fever, and/or being more proneto infections. Any combination of these symptoms of acute GVHD may beprevented, managed, treated, and/or ameliorated using the compositionsand methods provided herein.

In other embodiments, the GVHD is chronic GVHD. Chronic GVHD can occurfrom about three months to about a year or longer after transplant.Chronic GVHD can be mild or severe, and generally includes symptomssimilar to those of acute GVHD. Chronic GVHD can affect the skin anddigestive system, including the liver but can also involve other organsand the immune system (e.g., making the patient more prone toinfections) and/or connective tissues. Symptoms of chronic skin GVHDinclude a rash, dry skin, tight skin, itchy skin, darkening of thecolour of the skin, thickening of the skin, and/or may affect hair(e.g., hair loss, turning grey) or nails (e.g., hard or brittle nails).Chronic gut GVHD can affect the digestive system, mouth, oesophagus,lining of the stomach, and/or lining of the bowel, and symptoms caninclude diarrhoea, dry or sore mouth, painful swallowing, low nutrientabsorption by the stomach, bloating, stomach cramps. Chronic liver GVHDcan cause damage and scarring of the liver (cirrhosis). Chronic GVHD ofthe eyes can affect the glands that make tears, causing eyes to becomedry, burning and painful or difficult to tolerate bright light. Chroniclung GVHD can cause shortness of breath, wheezing, persistent cough,and/or being more prone to chest infections. Chronic GVHD affectstendons (e.g., inflammation) that connect muscle to bone causingdifficulty straightening or bending your arms and legs. Any combinationof these symptoms of chronic GVHD may be prevented, managed, treated,and/or ameliorated using the compositions and methods provided herein.

In certain embodiments provided herein are compositions comprising oneor more antibodies of the invention for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated disease,such as uveitis, or a symptom thereof.

In certain embodiments provided herein are compositions comprising oneor more antibodies of the invention for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated disease,such as pyoderma gangrenosum, giant cell arteritis, Schnitzler syndromeor non-infectious scleritis.

In certain embodiments provided herein are compositions comprising oneor more antibodies of the invention for use in the prevention,management, treatment and/or amelioration of a hOX40L mediated diseaseor condition selected from an autoimmunc disease or condition, asystemic inflammatory disease or condition, or transplant rejection; forexample inflammatory bowel disease (IBD), Crohn's disease, rheumatoidarthritis, transplant rejection, allogeneic transplant rejection,graft-versus-host disease (GvHD), ulcerative colitis, systemic lupuserythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contacthypersensitivity, multiple sclerosis and atherosclerosis, in particularGvHD.

In a specific embodiment, a composition for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated diseasecomprises the OX40L binding sites of an antibody of the invention, e.g.,an antibody disclosed in the Examples.

In another embodiment, a composition for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated diseasecomprises one or more antibodies comprising one or more VH domainshaving an amino acid sequence of any one of the VH domains in thesequence listing (i.e. Seq ID No:2, Seq ID No:34, Seq ID No:66 or Seq IDNo:94, in particular Seq ID No:34). In another embodiment, a compositionfor use in the prevention, management, treatment and/or amelioration ofa hOX40L-mediated disease comprises one or more antibodies comprisingone or more VH CDR1s having an amino acid sequence of any one of the VHCDR1s in the sequence listing (i.e. Seq ID No:4, Seq ID No:10, Seq IDNo:36, Seq ID No:42, Seq ID No:68, Seq ID No:74, Seq ID No:96 or Seq IDNo:102, in particular, Seq ID No:36 or Seq ID No:42). In anotherembodiment, a composition for use in the prevention, management,treatment and/or amelioration of a hOX40L-mediated disease comprises oneor more antibodies comprising one or more VH CDR2s having an amino acidsequence of any one of the VH CDR2s in the sequence listing (i.e. Seq IDNo:6, Seq ID No:12, Seq ID No:38, Seq ID No:44, Seq ID No:70, Seq IDNo:76, Seq ID No:98 or Seq ID No:104, in particular Seq ID No:38 or SeqID No:44). In a preferred embodiment, a composition for use in theprevention, management, treatment and/or amelioration of ahOX40L-mediated disease comprises one or more antibodies comprising oneor more VH CDR3s having an amino acid sequence of any one of the VHCDR3s in the sequence listing (i.e. Seq ID No:8, Seq ID No:14, Seq IDNo:40, Seq ID No:46, Seq ID No:72, Seq ID No:78, Seq ID No:100 or Seq IDNo:106, in particular Seq ID No:40 or Seq ID No:46).

In another embodiment, a composition for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated diseasecomprises one or more antibodies comprising one or more VL domainshaving an amino acid sequence of any one of the VL domains in thesequence listing (i.e. Seq ID No:16, Seq ID No:48, Seq ID No:80, or SeqID No:108, in particular Seq ID No:48) (optionally comprising also thecognate VH domain as set out in the sequence listing (i.e. Seq IDNo:2/16, Seq ID No:34/48, Seq ID No:66/80 or Seq ID No:94/108, inparticular Seq ID No:34/48). In another embodiment, a composition foruse in the prevention, management, treatment and/or amelioration of ahOX40L-mediated disease comprises one or more antibodies comprising oneor more VL CDR1s having an amino acid sequence of any one of the VLCDR1s in the sequence listing (i.e. Seq ID No:18, Seq ID No:24, Seq IDNo:50, Seq ID No:56, Seq ID No:82, Seq ID No:88, Seq ID No:110 or Seq IDNo:116, in particular Seq ID No:50 or Seq ID No:56). In anotherembodiment, a composition for use in the prevention, management,treatment and/or amelioration of a hOX40L-mediated disease comprises oneor more antibodies comprising one or more VL CDR2s having an amino acidsequence of any one of the VL CDR2s in the sequence listing (i.e. Seq IDNo:20, Seq ID No:26, Seq ID No:52, Seq ID No:58, Seq ID No:84, Seq IDNo:90, Seq ID No:112 or Seq ID No:118, in particular Seq ID No:52 or SeqID No:58). In a preferred embodiment, a composition for use in theprevention, management, treatment and/or amelioration of ahOX40L-mediated disease comprises one or more antibodies comprising oneor more VL CDR3s having an amino acid sequence of any one of the VLCDR3s in the sequence listing (i.e. Seq ID No:22, Seq ID No:28, Seq IDNo:54, Seq ID No:60, Seq ID No:86, Seq ID No:92, Seq ID No:114 or Seq IDNo:120, in particular Seq ID No:54 or Seq ID No:60).

In another embodiment, a composition for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated diseasecomprises one or more antibodies comprising one or more VH domainshaving an amino acid sequence of any one of the VH domains in thesequence listing (i.e. Seq ID No:2, Seq ID No:34, Seq ID No:66 or Seq IDNo:94, in particular Seq ID No:34), and one or more VL domains having anamino acid sequence of any one of the VL domains in the sequence listing(i.e. Seq ID No:16, Seq ID No:48, Seq ID No:80, or Seq ID No:108, inparticular Seq ID No:48).

In another embodiment, a composition for use in the prevention,management, treatment and/or amelioration of a hOX40L-mediated diseasecomprises one or more antibodies comprising one or more VH CDR1s havingan amino acid sequence of any one of the VH CDR1s in the sequencelisting (i.e. Seq ID No:4, Seq ID No:10, Seq ID No:36, Seq ID No:42, SeqID No:68, Seq ID No:74, Seq ID No:96 or Seq ID No:102, in particular,Seq ID No:36 or Seq ID No:42), and one or more VL CDR1s having an aminoacid sequence of any one of the VL CDR1s in the sequence listing (i.e.Seq ID No:18, Seq ID No:24, Seq ID No:50, Seq ID No:56, Seq ID No:82,Seq ID No:88, Seq ID No:110 or Seq ID No:116, in particular Seq ID No:50or Seq ID No:56). In another embodiment, a composition for use in theprevention, management, treatment and/or amelioration of ahOX40L-mediated disease comprises one or more antibodies comprising oneor more VH CDR1s having an amino acid sequence of any one of the VHCDR1s in the sequence listing (i.e. Seq ID No:4, Seq ID No:10, Seq IDNo:36, Seq ID No:42, Seq ID No:68, Seq ID No:74, Seq ID No:96 or Seq IDNo:102, in particular, Seq ID No:36 or Seq ID No:42), and one or more VLCDR2s having an amino acid sequence of any one of the VL CDR2s in thesequence listing (i.e. Seq ID No:20, Seq ID No:26, Seq ID No:52, Seq IDNo:58, Seq ID No:84, Seq ID No:90, Seq ID No:112 or Seq ID No:118, inparticular Seq ID No:52 or Seq ID No:58). In another embodiment, acomposition for use in the prevention, management, treatment and/oramelioration of a hOX40L-mediated disease comprises one or moreantibodies comprising one or more VH CDR1s having an amino acid sequenceof any one of the VH CDR1s in the sequence listing (i.e. Seq ID No:4,Seq ID No:10, Seq ID No:36, Seq ID No:42, Seq ID No:68, Seq ID No:74,Seq ID No:96 or Seq ID No:102, in particular, Seq TD No:36 or Seq TDNo:42), and one or more VL CDR3s having an amino acid sequence of anyone of the VL CDR3s having an amino acid sequence of any one of the VLCDR3s in the sequence listing (i.e. Seq ID No:22, Seq ID No:28, Seq IDNo:54, Seq ID No:60, Seq ID No:86, Seq ID No:92, Seq ID No:114 or Seq IDNo:120, in particular Seq ID No:54 or Seq ID No:60).

As discussed in more detail elsewhere herein, a composition of theinvention may be used either alone or in combination with othercompounds or compositions. Moreover, the antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

In some embodiments, provided herein are methods for decreasing orinhibiting binding of hOX40L to an OX40L receptor or cognate ligand(e.g., OX40) in a subject (e.g., a human subject), comprisingadministering to the subject an effective amount of an antibody thatspecifically binds to a hOX40L polypeptide (e.g., a cellsurface-expressed or soluble hOX40L). In some embodiments, a hOX40Lbiological activity, such as secretion of CCL20, TL8 and/or RANTES, orINF-γ, TNF-α or IL-2, in particular INF-γ or another cytokine disclosedherein, is also decreased in the subject, for example decreased by atleast 10, 20, 30, 40, 50 or 60%, or 70%, or 80%, or 90% or 95% or >95%.

In certain embodiments, provided herein are methods for decreasing orinhibiting a hOX40L biological activity, such as secretion of interferongamma, IL-2, CCL20, 1L8 and/or RANTES or other cytokine, or INF-γ, TNF-αor IL-2, in particular INF-γ in a subject (e.g., a human subject),comprising administering to the subject an effective amount of anantibody that specifically binds to a hOX40L polypeptide (e.g., a cellsurface-expressed hOX40L), wherein hOX40L biological activity isdecreased by the antibody.

In other embodiments, provided herein are methods for decreasing orinhibiting binding of hOX40L to an OX40L receptor or cognate ligand(e.g., OX40) in a cell having cell surface-expressed hOX40L, contactingthe cell with an effective amount of an antibody that specifically bindsto a hOX40L polypeptide (e.g., a cell surface-expressed or solublehOX40L), such as a hOX40L polypeptide, a hOX40L polypeptide fragment, ora hOX40L epitope. In some embodiments, a hOX40L biological activity,such as secretion of interferon gamma, IL-2, CCL20, IL8 and/or RANTES,or INF-γ, TNF-α or IL-2, in particular INF-γ or other cytokine disclosedherein, is also decreased in the cell.

In certain embodiments, provided herein are methods for decreasing orinhibiting a hOX40L biological activity, such as secretion of interferongamma, IL-2, CCL20, IL8 and/or RANTES or other cytokine disclosedherein, in a cell having a cell surface-expressed hOX40L receptor (suchas OX40), contacting the cell with an effective amount of an antibodythat specifically binds to a hOX40L polypeptide (e.g., a cellsurface-expressed or soluble hOX40L) wherein hOX40L biological activityis decreased by the antibody.

Antibodies of the present invention may be used, for example, to purify,detect, and target hOX40L antigens, in both in vitro and in vivodiagnostic and therapeutic methods. For example, the modified antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of hOX40L in biological samples. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988) (incorporated by reference herein in its entirety).

The invention also provides methods of preventing, managing, treatingand/or ameliorating a hOX40L-mediated disease by administrating to asubject of an effective amount of an antibody, or pharmaceuticalcomposition comprising an antibody of the invention. In one aspect, anantibody is substantially purified (i.e., substantially free fromsubstances that limit its effect or produce undesired side-effects). Inpreferred embodiments, the antibody is a fully human monoclonalantibody, such as a fully human monoclonal antagonist antibody. Thesubject administered a therapy is preferably a mammal such asnon-primate (e.g., cows, pigs, horses, cats, dogs, rodents, mice orrats) or a primate (e.g., a monkey, such as a rhesus or cynomolgousmonkey, or a human). In a preferred embodiment, the subject is a human.In another preferred embodiment, the subject is a human infant or ahuman infant born prematurely. In another embodiment, the subject is ahuman with a hOX40L-mediated disease.

Various delivery systems are known and can be used to administer aprophylactic or therapeutic agent (e.g., an antibody of the invention),including, but not limited to, encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe antibody, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of administering aprophylactic or therapeutic agent (e.g., an antibody of the invention),or pharmaceutical composition include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal and oral routes). In a specific embodiment, aprophylactic or therapeutic agent (e.g., an antibody of the presentinvention), or a pharmaceutical composition is administeredintranasally, intramuscularly, intravenously, or subcutaneously. Theprophylactic or therapeutic agents or compositions may be administeredby any convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, intranasal mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, eachof which is incorporated herein by reference their entirety.

In a specific embodiment, it may be desirable to administer aprophylactic or therapeutic agent, or a pharmaceutical composition ofthe invention locally to the area in need of treatment. This may beachieved by, for example, and not by way of limitation, local infusion,by topical administration (e.g., by intranasal spray), by injection, orby means of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering an antibody of the invention,care must be taken to use materials to which the antibody does notabsorb.

In another embodiment, a prophylactic or therapeutic agent, or acomposition of the invention can be delivered in a vesicle, inparticular a liposome (see Langer, 1990, Science 249:1527-1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In another embodiment, a prophylactic or therapeutic agent, or acomposition of the invention can be delivered in a controlled release orsustained release system. In one embodiment, a pump may be used toachieve controlled or sustained release (see Langer, supra; Sefton,1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In anotherembodiment, polymeric materials can be used to achieve controlled orsustained release of a prophylactic or therapeutic agent (e.g., anantibodies of the invention) or a composition of the invention (seee.g., Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat. No.5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat.No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No.WO 99/20253. Examples of polymers used in sustained release formulationsinclude, but are not limited to, poly(2-hydroxy ethyl methacrylate),poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinylacetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. In a preferred embodiment, the polymer usedin a sustained release formulation is inert, free of leachableimpurities, stable on storage, sterile, and biodegradable. In yetanother embodiment, a controlled or sustained release system can beplaced in proximity of the therapeutic target, i.e., the nasal passagesor lungs, thus requiring only a fraction of the systemic dose (see,e.g., Goodson, in Medical Applications of Controlled Release, supra,vol. 2, pp. 115-138 (1984)). Controlled release systems are discussed inthe review by Langer (1990, Science 249:1527-1533). Any technique knownto one of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies of the invention. See,e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCTpublication WO 96/20698, Ning et al., 1996, “IntratumoralRadioimmunotherapy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.,1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek etal., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854, and Lam et al., 1997, “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Intl. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in their entirety.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent (e.g., anantibody of the invention), the nucleic acid can be administered in vivoto promote expression of its encoded prophylactic or therapeutic agent,by constructing it as part of an appropriate nucleic acid expressionvector and administering it so that it becomes intracellular, e.g., byuse of a retroviral vector (see U.S. Pat. No. 4,980,286), or by directinjection, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see, e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

In a specific embodiment, a composition of the invention comprises one,two or more antibodies or fragments of the invention. In anotherembodiment, a composition of the invention comprises one, two or moreantibodies or fragments of the invention and a prophylactic ortherapeutic agent other than an antibody of the invention. Preferably,the agents are known to be useful for or have been or are currently usedfor the prevention, management, treatment and/or amelioration of ahOX40L-mediated disease. In addition to prophylactic or therapeuticagents, the compositions of the invention may also comprise a carrier.

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., compositionsthat are suitable for administration to a subject or patient) that canbe used in the preparation of unit dosage forms. In a preferredembodiment, a composition of the invention is a pharmaceuticalcomposition. Such compositions comprise a prophylactically ortherapeutically effective amount of one or more prophylactic ortherapeutic agents (e.g., an antibody of the invention or otherprophylactic or therapeutic agent), and a pharmaceutically acceptablecarrier. Preferably, the pharmaceutical compositions are formulated tobe suitable for the route of administration to a subject.

In a specific embodiment, the term “carrier” refers to a diluent,adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient,or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa. Suchcompositions will contain a prophylactically or therapeuticallyeffective amount of the antibody, preferably in purified form, togetherwith a suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anaesthetic such as lignocamne to ease pain at thesite of the injection. Such compositions, however, may be administeredby a route other than intravenous.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The invention also provides that an antibody of the invention ispackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity of antibody. In one embodiment, theantibody is supplied as a dry sterilized lyophilized powder or waterfree concentrate in a hermetically sealed container and can bereconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. Preferably, the antibodyis supplied as a dry sterile lyophilized powder in a hermetically sealedcontainer at a unit dosage of at least 0.1 mg, at least 0.5 mg, at least1 mg, at least 2 mg, or at least 3 mg, and more preferably at least 5mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, atleast 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, orat least 100 mg. The lyophilized antibody can be stored at between 2 and8° C. in its original container and the antibody can be administeredwithin 12 hours, preferably within 6 hours, within 5 hours, within 3hours, or within 1 hour after being reconstituted. In an alternativeembodiment, an antibody is supplied in liquid form in a hermeticallysealed container indicating the quantity and concentration of theantibody. Preferably, the liquid form of the antibody is supplied in ahermetically sealed container at least 0.1 mg/ml, at least 0.5 mg/ml, orat least 1 mg/ml, and more preferably at least 5 mg/ml, at least 10mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least40 mg/ml, at least 50 mg/ml, at least 60 mg/ml, at least 70 mg/ml, atleast 80 mg/ml, at least 90 mg/ml, or at least 100 mg/ml.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of a prophylactic or therapeutic agent (e.g., an antibody ofthe invention), or a composition of the invention that will be effectivein the prevention, management, treatment and/or amelioration of ahOX40L-mediated disease can be determined by standard clinicaltechniques.

Accordingly, a dosage of an antibody or a composition that results in aserum titer of from about 0.1 μg/ml to about 450 μg/ml, and in someembodiments at least 0.1 μg/ml, at least 0.2 μg/ml, at least 0.4 μg/ml,at least 0.5 μg/ml, at least 0.6 μg/ml, at least 0.8 μg/ml, at least 1μg/ml, at least 1.5 μg/ml, and preferably at least 2 μg/ml, at least 5μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least25 μg/ml, at least 30 μg/ml, at least 35 μg/ml, at least 40 μg/ml, atleast 50 μg/ml, at least 75 μg/ml, at least 100 μg/ml, at least 125μg/ml, at least 150 μg/ml, at least 200 μg/ml, at least 250 μg/ml, atleast 300 μg/ml, at least 350 μg/ml, at least 400 μg/ml, or at least 450μg/ml can be administered to a human for the prevention, management,treatment and/or amelioration of a hOX40L-mediated disease. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of ahOX40L-mediated disease, and should be decided according to the judgmentof the practitioner and each patient's circumstances.

Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For the antibodies of the invention, the dosage administered to apatient is typically 0.1 mg/kg to 100 mg/kg of the patient's bodyweight. In some embodiments, the dosage administered to the patient isabout 1 mg/kg to about 75 mg/kg of the patient's body weight.Preferably, the dosage administered to a patient is between 1 mg/kg and20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 5mg/kg of the patient's body weight. Generally, human antibodies have alonger half-life within the human body than antibodies from otherspecies due to the immune response to the foreign polypeptides. Thus,lower dosages of human antibodies and less frequent administration isoften possible. Further, the dosage and frequency of administration ofthe antibodies of the invention may be reduced by enhancing uptake andtissue penetration of the antibodies by modifications such as, forexample, lipidation.

In one embodiment, approximately 100 mg/kg or less, approximately 75mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg orless, approximately 10 mg/kg or less, approximately 5 mg/kg or less,approximately 1 mg/kg or less, approximately 0.5 mg/kg or less, orapproximately 0.1 mg/kg or less of an antibody or fragment the inventionis administered 5 times, 4 times, 3 times, 2 times or, preferably, 1time to manage a hOX40L-mediated disease. In some embodiments, anantibody of the invention is administered about 1-12 times, wherein thedoses may be administered as necessary, e.g., weekly, biweekly, monthly,bimonthly, trimonthly, etc., as determined by a physician. In someembodiments, a lower dose (e.g., 1-15 mg/kg) can be administered morefrequently (e.g., 3-6 times). In other embodiments, a higher dose (e.g.,25-100 mg/kg) can be administered less frequently (e.g., 1-3 times).However, as will be apparent to those in the art, other dosing amountsand schedules are easily determinable and within the scope of theinvention.

In a specific embodiment, approximately 100 mg/kg, approximately 75mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg orless, approximately 10 mg/kg or less, approximately 5 mg/kg or less,approximately 1 mg/kg or less, approximately 0.5 mg/kg or less,approximately 0.1 mg/kg or less of an antibody or fragment the inventionin a sustained release formulation is administered to a subject,preferably a human, to prevent, manage, treat and/or ameliorate ahOX40L-mediated disease. In another specific embodiment, anapproximately 100 mg/kg, approximately 75 mg/kg or less, approximately50 mg/kg or less, approximately 25 mg/kg or less, approximately 10 mg/kgor less, approximately 5 mg/kg or less, approximately 1 mg/kg or less,approximately 0.5 mg/kg or less, or approximately 0.1 mg/kg or lessbolus of an antibody the invention not in a sustained releaseformulation is administered to a subject, preferably a human, toprevent, manage, treat and/or ameliorate a hOX40L-mediated disease, andafter a certain period of time, approximately 100 mg/kg, approximately75 mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kgor less, approximately 10 mg/kg or less, approximately 5 mg/kg or less,approximately 1 mg/kg or less, approximately 0.5 mg/kg or less, orapproximately 5 mg/kg or less of an antibody of the invention in asustained release is administered to said subject (e.g., intranasally orintramuscularly) two, three or four times (preferably one time). Inaccordance with this embodiment, a certain period of time can be 1 to 5days, a week, two weeks, or a month.

In some embodiments, a single dose of an antibody or fragment of theinvention is administered to a patient to prevent, manage, treat and/orameliorate a hOX40L-mediated disease two, three, four, five, six, seven,eight, nine, ten, eleven, twelve times, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two,twenty-three, twenty-four, twenty five, or twenty six at bi-weekly(e.g., about 14 day) intervals over the course of a year, wherein thedose is selected from the group consisting of about 0.1 mg/kg, about 0.5mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg,about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg,about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combinationthereof (i.e., each dose monthly dose may or may not be identical).

In another embodiment, a single dose of an antibody of the invention isadministered to patient to prevent, manage, treat and/or ameliorate ahOX40L-mediated disease two, three, four, five, six, seven, eight, nine,ten, eleven, or twelve times at about monthly (e.g., about 30 day)intervals over the course of a year, wherein the dose is selected fromthe group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg,about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg,about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg,about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., eachdose monthly dose may or may not be identical).

In one embodiment, a single dose of an antibody or fragment of theinvention is administered to a patient to prevent, manage, treat and/orameliorate a hOX40L-mediated disease two, three, four, five, or sixtimes at about bi-monthly (e.g., about 60 day) intervals over the courseof a year, wherein the dose is selected from the group consisting ofabout 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg,about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg,about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about100 mg/kg, or a combination thereof (i.e., each bi-monthly dose may ormay not be identical).

In some embodiments, a single dose of an antibody or fragment of theinvention is administered to a patient to prevent, manage, treat and/orameliorate a hOX40L-mediated disease two, three, or four times at abouttri-monthly (e.g., about 120 day) intervals over the course of a year,wherein the dose is selected from the group consisting of about 0.1mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg,about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg,about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg,or a combination thereof (i.e., each tri-monthly dose may or may not beidentical).

In certain embodiments, the route of administration for a dose of anantibody or fragment of the invention to a patient is intranasal,intramuscular, intravenous, or a combination thereof, but other routesdescribed herein are also acceptable. In certain embodiments, the routeof administration is intraocular. Each dose may or may not beadministered by an identical route of administration. In someembodiments, an antibody or fragment of the invention may beadministered via multiple routes of administration simultaneously orsubsequently to other doses of the same or a different antibody orfragment of the invention.

In certain embodiments, antibodies or fragments of the invention areadministered prophylactically or therapeutically to a subject.Antibodies or fragments of the invention can be prophylactically ortherapeutically administered to a subject so as to prevent, lessen orameliorate a hOX40L-mediated disease or symptom thereof.

Gene Therapy

In a specific embodiment, nucleic acids or nucleotide sequences of theinvention are administered to prevent, manage, treat and/or ameliorate ahOX40L-mediated disease by way of gene therapy. Gene therapy refers totherapy performed by the administration to a subject of an expressed orexpressible nucleic acid. In an embodiment of the invention, the nucleicacids produce their encoded antibody, and the antibody mediates aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention.

Diagnostic Use of Antibodies

Although antibodies are mentioned in respect of diagnostic uses, thisdisclosure is to be read as also applying mutatis mutandis to thefragments of the invention.

Labelled antibodies or of the invention and derivatives and analoguesthereof, which specifically bind to a hOX40L antigen can be used fordiagnostic purposes to detect, diagnose, or monitor a hOX40L-mediateddisease. The invention provides methods for the detection of ahOX40L-mediated disease comprising: (a) assaying the expression of ahOX40L antigen in cells or a tissue sample of a subject using one ormore antibodies of the invention that specifically bind to the hOX40Lantigen; and (b) comparing the level of the hOX40L antigen with acontrol level, e.g., levels in normal tissue samples (e.g., from apatient not having a hOX40L-mediated disease, or from the same patientbefore disease onset), whereby an increase in the assayed level ofhOX40L antigen compared to the control level of the hOX40L antigen isindicative of a hOX40L-mediated disease.

The invention provides a diagnostic assay for diagnosing ahOX40L-mediated disease comprising: (a) assaying for the level of ahOX40L antigen in cells or a tissue sample of an individual using one ormore antibodies of the invention that specifically bind to a hOX40Lantigen; and (b) comparing the level of the hOX40L antigen with acontrol level, e.g., levels in normal tissue samples, whereby anincrease in the assayed hOX40L antigen level compared to the controllevel of the hOX40L antigen is indicative of a hOX40L-mediated disease.A more definitive diagnosis of a hOX40L-mediated disease may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the hOX40L-mediated disease.

Antibodies of the invention can be used to assay hOX40L antigen levelsin a biological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al.,1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods usefulfor detecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹²⁵I, ¹²¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of ahOX40L-mediated disease in a human. In one embodiment, diagnosiscomprises: a) administering (for example, parenterally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labelledantibody that specifically binds to a hOX40L antigen; b) waiting for atime interval following the administering for permitting the labelledantibody to preferentially concentrate at sites in the subject where thehOX40L antigen is expressed (and for unbound labelled molecule to becleared to background level); c) determining background level; and d)detecting the labelled antibody in the subject, such that detection oflabelled antibody above the background level indicates that the subjecthas a hOX40L-mediated disease. Background level can be determined byvarious methods including, comparing the amount of labelled moleculedetected to a standard value previously determined for a particularsystem.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹Tc. The labelled antibody willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumour imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labelled antibody to preferentially concentrate atsites in the subject and for unbound labelled antibody to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of a hOX40L-mediated disease is carriedout by repeating the method for diagnosing the a hOX40L-mediateddisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

Presence of the labelled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labelled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labelled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labelled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labelledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Methods of Producing Antibodies

Antibodies and fragments of the invention that specifically bind to anantigen (OX40L) can be produced by any method known in the art for thesynthesis of antibodies, in particular, by chemical synthesis orpreferably, by recombinant expression techniques. The practice of theinvention employs, unless otherwise indicated, conventional techniquesin molecular biology, microbiology, genetic analysis, recombinant DNA,organic chemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described in the references citedherein and are fully explained in the literature. See, e.g., Maniatis etal. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press; Sambrook et al. (1989), Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press;Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons (1987 andannual updates); Current Protocols in Immunology, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Birren et al. (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

Polyclonal antibodies that specifically bind to an antigen can beproduced by various procedures well-known in the art. For example, ahuman antigen can be administered to various host animals including, butnot limited to, rabbits, mice, rats, etc. to induce the production ofsera containing polyclonal antibodies specific for the human antigen.Various adjuvants may be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminiumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and cornyebacterium parvum. Suchadjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas 563 681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. Other exemplary methods of producing monoclonal antibodiesare discussed elsewhere herein, such as e.g., use of the KM Mouse™.Additional exemplary methods of producing monoclonal antibodies areprovided in the Examples herein.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with a hOX40L antigen and once an immune responseis detected, e.g., antibodies specific for hOX40L antigen are detectedin the mouse serum, the mouse spleen is harvested and splenocytesisolated. The splenocytes are then fused by well known techniques to anysuitable myeloma cells, for example cells from cell line SP20 availablefrom the ATCC. Hybridomas are selected and cloned by limited dilution.

Additionally, a RIMMS (repetitive immunization multiple sites) techniquecan be used to immunize an animal (Kilptrack et al., 1997 Hybridoma16:381-9, incorporated by reference in its entirety). The hybridomaclones are then assayed by methods known in the art for cells thatsecrete antibodies capable of binding a polypeptide of the invention.Ascites fluid, which generally contains high levels of antibodies, canbe generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingantibodies by culturing a hybridoma cell secreting a modified antibodyof the invention wherein, preferably, the hybridoma is generated byfusing splenocytes isolated from a mouse immunized with a hOX40L antigenwith myeloma cells and then screening the hybridomas resulting from thefusion for hybridoma clones that secrete an antibody able to bind to ahOX40L antigen.

Antibody fragments which recognize specific hOX40L antigens may begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)₂ fragments of the invention may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the variable region, the Lightchain constant region and the CH1 domain of the heavy chain. Further,the antibodies of the present invention can also be generated usingvarious phage display methods known in the art.

For example, antibodies can also be generated using various phagedisplay methods. In phage display methods, functional antibody domainsare displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In particular, DNA sequencesencoding VH and VL domains are amplified from animal cDNA libraries(e.g., human or murine cDNA libraries of affected tissues). The DNAencoding the VH and VL domains are recombined together with an scFvlinker by PCR and cloned into a phagemid vector. The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to a particular antigen can be selected oridentified with antigen, e.g., using labelled antigen or antigen boundor captured to a solid surface or bead. Examples of phage displaymethods that can be used to make the antibodies of the present inventioninclude those disclosed in Brinkman et al., 1995, J. Immunol. Methods182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al.,1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology57:191-280; PCT Application No. PCT/GB91/01134; InternationalPublication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727,5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)₂ fragments can also be employed using methods knownin the art such as those disclosed in PCT publication No. WO 92/22324;Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995,AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043 (saidreferences incorporated by reference in their entireties).

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing a VHconstant region, e.g., the human gamma 4 constant region, and the PCRamplified VL domains can be cloned into vectors expressing a VL constantregion, e.g., human kappa or lambda constant regions. The VH and VLdomains may also cloned into one vector expressing the necessaryconstant regions. The heavy chain conversion vectors and light chainconversion vectors are then co-transfected into cell lines to generatestable or transient cell lines that express full-length antibodies,e.g., IgG, using techniques known to those of skill in the art.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use human or chimericantibodies. Completely human antibodies are particularly desirable fortherapeutic treatment of human subjects. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.

In preferred embodiments, human antibodies are produced. Humanantibodies and/or fully human antibodies can be produced using anymethod known in the art, including the Examples provided herein. Forexample, transgenic mice which are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes. For example, the human heavy and light chain immunoglobulin genecomplexes may be introduced randomly or by homologous recombination intomouse embryonic stem cells. Alternatively, the human variable region,constant region, and diversity region may be introduced into mouseembryonic stem cells in addition to the human heavy and light chaingenes. The mouse heavy and light chain immunoglobulin genes may berendered non-functional separately or simultaneously with theintroduction of human immunoglobulin loci by homologous recombination.In particular, homozygous deletion of the JH region prevents endogenousantibody production. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of a polypeptide of theinvention. Monoclonal antibodies directed against the antigen can beobtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B-cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publication Nos. WO98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923,5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and5,939,598, which are incorporated by reference herein in their entirety.Other methods are detailed in the Examples herein. In addition,companies such as Abgenix, Inc/Amgen. (Thousand Oaks, Calif.) OMT (PaoloAlto, Calif.), Argen-x (Breda, Netherlands), Ablexis (San Francisco,Calif.) or Harbour Antibodies (Cambridge, Mass.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Methodsfor producing chimeric antibodies are known in the art. See, e.g.,Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214;Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat.Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, which areincorporated herein by reference in their entirety.

A humanized antibody is an antibody or its variant or fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab)₂, Fabc, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence.Preferably, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Ordinarily, the antibody will contain both the lightchain as well as at least the variable domain of a heavy chain. Theantibody also may include the CHL hinge, CH2, CH3, and CH4 regions ofthe heavy chain The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. Usually the constant domain is acomplement fixing constant domain where it is desired that the humanizedantibody exhibit cytotoxic activity, and the class is typically IgG1.Where such cytotoxic activity is not desirable, the constant domain maybe of the IgG2 class. In certain embodiments, the antibodies of theinvention comprise a human gamma 4 constant region. In anotherembodiment, the heavy chain constant region does not bind Fc-γreceptors, and e.g. comprises a Leu235Glu mutation. In anotherembodiment, the heavy chain constant region comprises a Ser228Promutation to increase stability. In another embodiment, the heavy chainconstant region is IgG4-PE. Examples of VL and VH constant domains thatcan be used in certain embodiments of the invention include, but are notlimited to, C-kappa and C-gamma-1 (nGlm) described in Johnson et al.(1997) J. Infect. Dis. 176, 1215-1224 and those described in U.S. Pat.No. 5,824,307. The humanized antibody may comprise sequences from morethan one class or isotype, and selecting particular constant domains tooptimize desired effector functions is within the ordinary skill in theart. The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the importantibody. Such mutations, however, will not be extensive. Usually, atleast 75% of the humanized antibody residues will correspond to those ofthe parental FR and CDR sequences, more often 90%, and most preferablygreater than 95%. Humanized antibodies can be produced using variety oftechniques known in the art, including but not limited to, CDR-grafting(European Patent No. EP 239,400; International publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089),veneering or resurfacing (European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering 7(6):805-814; and Roguska et al.,1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No.5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119 25 (2002),Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84(1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al.,Cancer Res. 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res.55(8):1717-22 (1995), Sandhu J S, Gene 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S.Patent Pub. No. US 2005/0042664 A1 (Feb. 24, 2005), which isincorporated by reference herein in its entirety. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modelling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Reichmann et al., 1988, Nature332:323, which are incorporated herein by reference in theirentireties.)

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well-known in the art. See Riechmannet al., 1999, J. Immunol. 231:25-38; Nuttall et al., 2000, Curr. Pharm.Biotechnol. 1(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302;U.S. Pat. No. 6,005,079; and International Publication Nos. WO 94/04678,WO 94/25591, and WO 01/44301, each of which is incorporated herein byreference in its entirety.

Further, the antibodies that specifically bind to a hOX40L antigen can,in turn, be utilized to generate anti-idiotype antibodies that “mimic”an antigen using techniques well known to those skilled in the art.(See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; andNissinoff, 1991, J. Immunol., 147(8):2429-2438).

Kits

The invention also provides a pharmaceutical or diagnostic pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention, such asone or more antibodies or fragments provided herein. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration,e.g., an authorisation number.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated hOX40L antigen as a control. Preferably, the kitsof the present invention further comprise a control antibody which doesnot react with the hOX40L antigen. In another specific embodiment, thekits of the present invention contain a means for detecting the bindingof a modified antibody to a hOX40L antigen (e.g., the antibody may beconjugated to a detectable substrate such as a fluorescent compound, anenzymatic substrate, a radioactive compound or a luminescent compound,or a second antibody which recognizes the first antibody may beconjugated to a detectable substrate). In specific embodiments, the kitmay include a recombinantly produced or chemically synthesized hOX40Lantigen. The hOX40L antigen provided in the kit may also be attached toa solid support. In a more specific embodiment the detecting means ofthe above described kit includes a solid support to which hOX40L antigenis attached. Such a kit may also include a non-attachedreporter-labelled anti-human antibody. In this embodiment, binding ofthe antibody to the hOX40L antigen can be detected by binding of thesaid reporter-labelled antibody.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the blood brainbarrier (BBB)). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.Mol Biol, 1999, 217, 721-739 and Taylor et al., J. Theor. Biol.119(1986); 205-218 and S. French and B. Robson, J. Mol. Evol.,19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpeptide (i.e. amino acids exposed to a solvent), for example, but notlimited to, the following substitutions can be used: substitution of Ywith F, T with S or K, P with A, E with D or Q, N with D or G, R with K,G with N or A, T with S or K, D with N or E, I with L or V, F with Y, Swith T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulphide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria. Antibodies can be humanized usingroutine technology.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody fragment” refers to a polypeptidethat includes at least one immunoglobulin variable domain orimmunoglobulin variable domain sequence and which specifically binds agiven antigen. An antibody fragment can comprise an antibody or apolypeptide comprising an antigen-binding domain of an antibody. In someembodiments, an antibody fragment can comprise a monoclonal antibody ora polypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and an OX40L (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two OX40L (L)chain variable regions. The term “antibody fragment” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments (sec, e.g. de Wildt et al., Eur J.Immunol., 1996; 26(3):629-39; which is incorporated by reference hereinin its entirety)) as well as complete antibodies. An antibody can havethe structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesand combinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

As used herein, “antibody variable domain” refers to the portions of theOX40L and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; i.e., CDR1,CDR2, and CDR3), and Framework Regions (FRs). VH refers to the variabledomain of the heavy chain VL refers to the variable domain of the Lightchain. According to the methods used in this invention, the amino acidpositions assigned to CDRs and FRs may be defined according to Kabat(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991)) or according to IMGTnomenclature.

As used herein, the term “antibody binding site” refers to a polypeptideor domain that comprises one or more CDRs of an antibody and is capableof binding an antigen. For example, the polypeptide comprises a CDR3(e.g., HCDR3). For example the polypeptide comprises CDRs 1 and 2 (e.g.,HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (e.g.,HCDRs 1-3). In an example, the antibody binding site is provided by asingle variable domain (e.g., a VH or VL domain). In another example,the binding site comprises a VH/VL pair or two or more of such pairs.

As used herein, “genotyping” refers to a process of determining thespecific allelic composition of a cell and/or subject at one or moreposition within the genome, e.g. by determining the nucleic acidsequence at that position. Genotyping refers to a nucleic acid analysisand/or analysis at the nucleic acid level. As used herein, “phenotyping”refers a process of determining the identity and/or composition of anexpression product of a cell and/or subject, e.g. by determining thepolypeptide sequence of an expression product. Phenotyping refers to aprotein analysis and/or analysis at the protein level.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment). For treatment to beeffective a complete cure is not contemplated. The method can in certainaspects include cure as well.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

Multiple compositions can be administered separately or simultaneously.Separate administration refers to the two compositions beingadministered at different times, e.g. at least 10, 20, 30, or 10-60minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One canalso administer compositions at 24 hours apart, or even longer apart.Alternatively, two or more compositions can be administeredsimultaneously, e.g. less than 10 or less than 5 minutes apart.Compositions administered simultaneously can, in some aspects, beadministered as a mixture, with or without similar or different timerelease mechanism for each of the components.

As used herein, “authorization number” or “marketing authorizationnumber” refers to a number issued by a regulatory agency upon thatagency determining that a particular medical product and/or compositionmay be marketed and/or offered for sale in the area under the agency'sjurisdiction. As used herein “regulatory agency” refers to one of theagencies responsible for evaluating, e.g., the safety and efficacy of amedical product and/or composition and controlling the sales/marketingof such products and/or compositions in a given area. The Food and DrugAdministration (FDA) in the US and the European Medicines Agency (EPA)in Europe are but two examples of such regulatory agencies. Othernon-limiting examples can include SDA, MPA, MHPRA, IMA, ANMAT, Hong KongDepartment of Health-Drug Office, CDSCO, Medsafe, and KFDA.

As used herein, “injection device” refers to a device that is designedfor carrying out injections, an injection including the steps oftemporarily fluidically coupling the injection device to a person'stissue, typically the subcutaneous tissue. An injection further includesadministering an amount of liquid drug into the tissue and decoupling orremoving the injection device from the tissue. In some embodiments, aninjection device can be an intravenous device or IV device, which is atype of injection device used when the target tissue is the blood withinthe circulatory system, e.g., the blood in a vein. A common, butnon-limiting example of an injection device is a needle and syringe.

As used herein, a “buffer” refers to a chemical agent that is able toabsorb a certain quantity of acid or base without undergoing a strongvariation in pH.

As used herein, “packaging” refers to how the components are organizedand/or restrained into a unit fit for distribution and/or use. Packagingcan include, e.g., boxes, bags, syringes, ampoules, vials, tubes,clamshell packaging, barriers and/or containers to maintain sterility,labeling, etc.

As used herein, “instructions” refers to a display of written, printedor graphic matter on the immediate container of an article, for examplethe written material displayed on a vial containing a pharmaceuticallyactive agent, or details on the composition and use of a product ofinterest included in a kit containing a composition of interest.Instructions set forth the method of the treatment as contemplated to beadministered or performed.

As used herein the term “comprising” or “comprises” is used in referenceto antibodies, fragments, uses, compositions, methods, and respectivecomponent(s) thereof, that are essential to the method or composition,yet open to the inclusion of unspecified elements, whether essential ornot.

The term “consisting of” refers to antibodies, fragments, uses,compositions, methods, and respective components thereof as describedherein, which are exclusive of any element not recited in thatdescription of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino etal. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in OX40L of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

It will be understood that particular configurations, concepts, aspects,examples, clauses and embodiments described herein are shown by way ofillustration and not as limitations of the invention. The principalfeatures of this invention can be employed in various embodimentswithout departing from the scope of the invention. Those skilled in theart will recognize, or be able to ascertain using no more than routinestudy, numerous equivalents to the specific procedures described herein.Such equivalents are considered to be within the scope of this inventionand are covered by the claims. All publications and patent applicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this invention pertains. Allpublications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” The use ofthe term “or” in the claims is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variationof error for the device, the method being employed to determine thevalue, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in OX40L of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

EXAMPLES Example 1

Antigen Preparation, Immunization Procedures, and Hybridoma Generation

The following example provides a detailed description of the generationand identification of a panel of anti-human OX40L monoclonal antibodiesusing the KyMouse™ system (see, e.g., WO2011/004192). To this end,genetically engineered mice containing a large number of humanimmunoglobulin genes were immunized with soluble recombinant human OX40L(commercial or in-house produced) or surface expressed human OX40Ldisplayed on mouse embryonic fibroblast (MEF) cells. Variousimmunization regimes, including conventional intraperitoneal injectionsas well as a rapid immunisation at multiple sites regime were set up,boosting animals over several weeks. At the end of each regime,secondary lymphoid tissue such as the spleen, and in some cases, thelymph nodes were removed. Tissues were prepared into a single cellsuspension and fused with SP2/0 cells to generate a stable hybridomacell line.

Materials and Methods Cloning Expression and Purification of RecombinantRhesus and Human OX40L

cDNA encoding the extracellular domain of human OX40L was cloned into apREP4 expression plasmid (Invitrogen) using standard molecular biologytechniques. The constructs also contained a FLAG peptide motif to aidpurification and an isoleucine zipper motif to aid trimerisation.Constructs were sequenced to ensure their correct sequence composition.

Rhesus (Macaca mulatta) OX40L was created using the human OX40L plasmidcreated above as a template and using site directed mutagenesis tointroduce the amino acid changes.

Human OX40L well as Rhesus monkey OX40L were expressed transiently toproduce recombinant protein using Invitrogen's FreeStyle™ CHO-Ssuspension adapted cell line. Plasmids were transfected into the cellsusing PEI (polyethylenimine 1\4W 40000) and left to overgrow for aperiod of 13 days before harvesting the supernatant for purification.Cells were fed during the overgrow process with ActiCHO™ Feeds A and Bfrom GE Healthcare to help boost productivity and promote longevity ofthe cells. During the overgrow process samples were taken regularly tomonitor cell growth and viability.

FLAG-tagged OX40L proteins were purified in a two-step process; firstlythe clarified tissue culture supernatants from the CHO-S expression werepurified using M2 anti-FLAG affinity chromatography. The elutedfractions containing the OX40L protein were then subjected to sizeexclusion chromatography and assessed for purity by SDS-PAGE analysisand quantified by spectrophotometer reading at OD280 nm.

Cloning Expression and Purification of Recombinant Human OX40 Receptor

cDNA encoding the extracellular domain of human OX40 Receptor was clonedinto a pREP4 expression plasmid (Invitrogen) using standard restrictionenzyme digestion and ligation. The construct contained a human Fcportion to aid purification. Constructs were sequenced to ensure theircorrect sequence composition.

Human OX40 Receptor was expressed transiently to produce recombinantprotein using Invitrogen's FreeStyle™ CHO-S suspension adapted cellline. Plasmids were transfected into the cells using PET(polyethylenimine MW 40000) and left to overgrow for a period of 13 daysbefore harvesting the supernatant for purification. Cells were fedduring the overgrow process with ActiCHO™ Feeds A and B from GEHealthcare to help boost productivity and promote longevity of thecells. During the overgrow process, samples were taken regularly tomonitor cell growth and viability.

The Fc tagged OX40 Receptor protein was purified in a two-step process;firstly the clarified tissue culture supernatants from the CHO-Sexpression were purified using Protein G affinity chromatography. Theeluted fractions containing the OX40 Receptor protein were thensubjected to size exclusion chromatography and assessed for purity bySDS-PAGE analysis and quantified by spectrophotometer reading at OD280nm.

Generation of Stably Transfected MEF and CHO-S Cells Expressing HumanOX40L

The full human OX40L sequences were codon optimized (Seq ID No:173) formammalian expression and cloned into an expression vector under the CMVpromoter flanked by 3′ and 5′ piggyBac specific terminal repeatsequences facilitating stable integration into the cell genome (see: “Ahyperactive piggyBac transposase for mammalian applications”; Yusa K,Zhou L, Li M A, Bradley A, Craig N L. Proc Natl Acad Sci USA. 2011 Jan.25). Furthermore, the expression vector contained either a puromycin orneomycin selection cassette to facilitate stable cell line generation.The hOX40L expression plasmid was co-transfected with a plasmid encodingpiggyBac transposase into an in-house derived mouse embryonic fibroblast(MEF) cell line (embryos used to generate this line were obtained from a129S5 crossed to C57BL6 female mouse) and CHO-S cells using theFreeStyle Max transfection reagent (Invitrogen) according tomanufacturer instructions. 24 hours after transfection, the media wassupplemented with G418 or neomycin and grown for at least 2 weeks toselect a stable cell line, with media being exchanged every 3-4 days.The expression of hOX40L was assessed by flow cytometry using ananti-human OX40L-PE conjugated antibody (eBioscience). Complete MEFmedia was made up of Dulbecco's Modified Eagle's Medium (Gibco)supplemented with 10% v/v fetal bovine scrum (Gibco). Complete CHO-Smedia was made up of CD-CHO media supplemented with 8 mM glutamax(Gibco).

Generation of HT1080 Expressing OX40R and NF-Kappa Reporter Gene

The full human OX40 receptor sequence was codon optimized (Seq TDNo:175) for mammalian expression and cloned into an expression vectorunder the CMV promoter flanked by 3′ and 5′ piggyBac specific terminalrepeat sequences facilitating stable integration into the cell genome(see: “A hyperactive piggyBac transposase for mammalian applications”;Yusa K, Zhou L, Li M A, Bradley A, Craig N L. Proc Natl Acad Sci USA.2011 Jan. 25). Furthermore, the expression vector contained either apuromycin selection cassette to facilitate stable cell line generation.The hOX40 receptor expression plasmid was co-transfected with a plasmidencoding piggyBac transposase into HT1080 cells (ATCC® CCL-121) usingthe FreeStyle Max transfection reagent (Invitrogen) according tomanufacturer instructions. 24 hours after transfection, the media wassupplemented with puromycin and grown for at least 2 weeks to select astable cell line with media being exchanged every 3-4 days. Theexpression of OX40 receptor was assessed by flow cytometry using ananti-human OX40 receptor-PE conjugated antibody (R&D, clone 443318).Following the generation of a stable cell line expressing the OX40receptor, cells were transfected with the pNiFty-2-SEAP plasmid(invivogen) containing 5 repeated NFkB transcription factor bindingsites followed by secreted alkaline phosphatase. Stable cells wereselected with the addition to zeocin to the media with fresh media beingadded every 3-4 days. Complete HT1080 media was made up of MEMsupplemented with 10% fetal calf serum.

Preparation of MEF Cells for Mouse Immunizations:

Cell culture medium was removed and cells washed once with 1×PBS. Cellswere treated for 5 minutes with trypsin to loosen cells from tissueculture surface. Cells were collected and trypsin neutralized by theaddition of complete media containing 10% v/v fetal bovine serum (FCS).Cells were then centrifuged at 300×g for 10 minutes and washed with 25mL of 1×PBS. Cells were counted and resuspended at the appropriateconcentration in 1×PBS.

Immunization Procedure:

Transgenic Kymice were immunized with hOX40L in either solublerecombinant form, expressed by CHO-S cells, or membrane bound form,expressed by stably transfected MEF cells.

When immunizing with cells, the adjuvant was mixed with cells at a 1:1v/v ratio and gently mixed by pipetting before injectingintraperitoneally. When immunizing with protein, the adjuvant was mixedwith protein at a 1:1 v/v ratio and vortexed repeatedly. All mice werebled before being primed and then boosted every three weeks. At least 3serial bleeds spaced apart at least 2 weeks were collected and analysedfor hOX40L specific IgG titre using an ELISA or flow cytometry basedassay

Determination of Serum Titers by FACS Using CHO-S Expressed hOX40L

CHO-S cells expressing hOX40L or untransfected CHO-S cells, diluted inFACS buffer (PBS+1% w/v BSA+0.1% w/v NaN₃) were distributed to a 96 wellV-bottom plate (Greiner) at a density of 1×10⁵ cells per well. Cellswere washed with 150 μl, of PBS and centrifuged at 300×g for 3 min.Supernatant was aspirated and 150 μL of PBS added. This wash step wasrepeated. A titration of mouse serum was prepared, diluting samples inFACS buffer. 50 μL/well of this titration was then added to the cellplate. To determine the change in activity level due to immunization,serum from each animal prior to immunization was diluted to 1 in 100 inFACS buffer and 50 μL/well added to the cells. A suitable referenceantibody (anti-OX40L antibody MAB10541, R&D systems) or mouse IgG1control antibody (Sigma) were diluted in FACS buffer (between 1-9 μg/mL)and 50 μL added to cells. Cells were incubated at 4° C. for 30 minutes.Cells were washed twice with 150 μL of PBS, centrifuging after each washstep and aspirating supernatant (centrifuged at 300×g for 3 minutes). Todetect antibody binding, APC goat-anti-mouse IgG (JacksonImmunoResearch) was diluted 1 in 500 in FACS buffer and 50 μL was addedto the cells. Cells were incubated 30 minutes at 4° C. in dark. Cellswere washed twice with 150 μL of PBS centrifuging after each wash stepand aspirating supernatant (centrifuged at 300×g for 3 minutes). To fixcells 100 μL 2% v/v paraformaldehyde was added and cells incubated for30 minutes at 4° C., cells were pelleted by centrifugation at 300×g andthe plates resuspended in 50 μL of FACS buffer. APC signal intensity(geomean) was measured by flow cytometry using a BD FACS Arrayinstrument.

Determination of Serum Titers by DELFIA Immunoassay Using RecombinanthOX40L

Titers in mouse serum samples were determined using a reverse OX40LELISA protocol. Anti-mouse IgG capture antibody (Southern Biotech) (4μg/mL diluted in PBS, 50 μL/well) was adsorbed to 96 well lowauto-fluorescent, high protein binding plates (Costar) overnight at 4°C. Excess IgG was removed by washing with PBS-Tween (0.1% v/v) and thewells were blocked with 1% w/v bovine serum albumin (BSA, Sigma) in PBSfor 1 hr at RT, after which plates were washed as described previously.A titration of mouse serum was prepared, diluting samples in reagentdiluent (0.1% w/v BSA/PBS). 50 μL/well of this titration was then addedto ELISA plates. To determine the change in activity level due toimmunization, serum from each animal prior to immunization was dilutedto 1 in 100 in reagent diluent and 50 μL/well added to the ELISA plate.As a positive control for biotinylated OX40L binding an anti-OX40Lantibody (MAB10541, R&D systems) diluted to 1 μg/mL was added to platesat 50 μL. Mouse IgG1 isotype control (Sigma) was included as a negativecontrol and was diluted to 1 μg/mL in reagent diluent and 50 μL/welladded to ELISA plate. In some instances serum sample from a mouseimmunized with a non-relevant antigen was diluted 1 in 1000 and 50μL/well was added to the ELISA plate. The plates were incubated at roomtemperature for at least 1 hour. Following incubation, plates werewashed as before to remove unbound proteins. Biotinylated OX40L (100ng/mL in reagent diluent; 50 μL/well) was then added to the plates andincubated at RT for 1 hour. Unbound biotinylated OX40L was removed bywashing with PBS-Tween (0.1% v/v), while the remaining biotinylatedOX40L was detected by streptavidin-Europium3+conjugate (DELFIA®detection, PerkinElmer) diluted in DELFIA® assay buffer (Perkin Elmer)or streptavidin-HRP diluted in reagent diluent.

In the case of streptavidin-HRP, the plates were washed as describedbefore and 50 μL of TMB (Sigma) was added to the plate. Then thereaction was stopped by adding 50 μL of 1M sulfuric acid (Flukaanalytical). The OD at 450 nm was measured on an Envision plate reader(PerkinElmer).

In case of streptavidin-Europium3, the plates were washed with TBS (Trisbuffered saline)-Tween (0.1% v/v) and 200 μL/well of DELFIA Enhancementsolution (Perkin Elmer) was added to the plate. The time-resolvedfluorescence was measured at 615 nm on an Envision plate reader(PerkinElmer). Fluorescence data was plotted as Europium counts.

Murine Tissue Isolation and Preparation:

Spleens were excised from immunised mice and washed in 1×PBS and kept onice until further processing. Tissues were prepared in buffer containing1×PBS (Invitrogen) and 3% heat-inactivated FBS (Invitrogen). Splenocyteswere dispersed by mashing the tissue through a 45 μm strainer (BDFalcon) and rinsing with 30 mL 3% FBS/PBS buffer before centrifugationat 700 g for 10 minutes at 4° C. To remove red blood cells, the pelletedsplenocytes were resuspended in 4 mL of Red Blood Cell Lysis Buffer(Sigma). After 4 minutes of incubation, the lysis reaction was stoppedby addition of 3% FBS/1×PBS buffer. Cell clumps were filtered out with a45 μm strainer. The remaining splenocytes were pelleted for furtherprocedures

Hybridoma Fusion

For the KM055 experiment, pelleted splenocytes were progressed directlyto fusion without any selection or overnight CpG stimulation. For theKM040 experiment, B-cells were subjected to a positive selection methodusing the MACS® Separation system. Cells were resuspended in 80 μL 3%FBS/PBS buffer per 1×10⁷ cells, before adding the anti-mouse IgG1 plusanti-mouse IgG2a+b MicroBeads (Miltenyi Biotec) and incubated for 15minutes at 4° C. The cells/MicroBeads mixture was then applied to apre-wetted LS column placed in a magnetic MACS Separator and washed with3% FBS/PBS buffer. IgG positive cells were collected in the labelled,column-bound fraction in 3% FBS/PBS buffer.

For the KM040 experiment, enriched B-cells were treated with CpGovernight (final concentration 25 μM) and the following day washed oncein BSA fusion buffer (0.3M D-Sorbitol, 0.11 mM calcium acetate hydrate,0.5 mM magnesium acetate tetrahydrate and 0.1% BSA (v/w), adjusted topH7.2). For the KM055 experiment, pelleted splenocytes from red bloodcell lysis were washed once in BSA fusion buffer on the same day astissue preparation. Fusion proceeded in the same way for bothexperiments after this point. Washed cells were resuspended in 200 μL ofBSA fusion buffer and cell count determined. SP2/0 cells were treated inthe same way, but washed twice with BSA fusion buffer. B-cells werefused at a ratio of 3:1 with SP2/0 myeloma cells by electrofusion usinga BTX ECM 2001 Electro Cell Manipulator (Harvard Apparatus). Each fusionwas left overnight in recovery medium (Dulbecco's Modified Eagle'sMedium—high glucose (no phenol red, no L-G) containing OPI (Sigma),L-Glutamax (Gibco), 20% FBS (Gibco, batch-tested for hybridoma) and2-mercaptoethanol). On the final day, cells were pelleted andresuspended in 1 part recovery medium to 9 parts semi-solid medium(ClonaCell-HY Hybridoma Selection Medium D, Stemcell Technologies) andthen seeded onto 10 cm petri dishes. Colonies were picked 12 days laterinto 96-well plates and cultured for another 2-3 days prior toscreening.

Example 2 Hybridoma Supernatant Screening

After generation of hybridoma clones, the hybridoma supernatant wasassessed in a sequential primary and secondary screen and appropriatehybridoma clones selected based on criteria of antibody binding to CHOexpressed hOX40L and receptor neutralization activity (see details inmaterials and methods) (Table 1).

For the primary screen, the inventors devised the following selectioncriteria: wells containing hybridoma clones were selected if antibodiespresent in the supernatant could bind to natively displayed hOX40Lexpressed on the cell surface. This assay was set up by plating CHO-Scells expressing hOX40L on the cell surface, followed by incubation withhybridoma supernatant, followed by a fluorescent detection antibody. Thepresence of an anti-OX40L antibody in the supernatant was read-out usinga plate reader capable of reading the appropriate fluorescence.Furthermore, the inventors assessed hybridoma supernatant for binding torecombinantly expressed human OX40L using an HTRF (Homogeneous TimeResolved Fluorescence) assay. The inventors also determined whether thehybridoma supernatant had the ability to reduce the binding of humanrecombinant OX40L to human OX40R Fc. Clones meeting certain selectioncriteria (see further detailed description below), using data from theabove mentioned three primary screen assays, were then cherry-picked andmoved on to a secondary screen where the ability of each antibody toneutralize hOX40L binding to its receptors, OX40 Receptor (aka CD134),was determined. The inventors decided to assess this using a receptorneutralization HTRF assay and a flow cytometry-based receptorneutralization assay. Lastly, the inventors decided to analyse hybridomasupernatant by SPR to evaluate apparent affinity of the antibodies torecombinant trimeric human OX40L as well as cross-reactivity to Rhesusmonkey OX40L.

Antibodies were defined as a secondary hit when antibodies in hybridomasupernatant bound to hOX40L, with high apparent affinity as well ascross-reacted with recombinant Rhesus monkey OX40L. Additionally,antibodies in the supernatant had to show the ability to neutralizeOX40L binding to its receptor, i.e. OX40 Receptor (aka CD134) in eitherHTRF or flow cytometry based assay.

Materials and Methods Primary Screen—Binding to Cell Expressed HumanOX40L

Supernatants collected from hybridoma cells were tested to assess theability of secreted antibodies to bind to hOX40L expressed on thesurface of CHO-S cells. To determine CHO-S hOX40L binding, cells wereplated in clear bottom tissue culture treated 384-well plates (Costar orBRAND) at 2×10⁴ cells/well in F12 media (GIBCO) supplemented with 10%v/v FBS (GIBCO) and cultured overnight. Culture media was removed from384-well assay plates. At least 40 μL of hybridoma supernatant orpositive control anti-human OX40L reference antibody (at a finalconcentration of 1 μg/mL) or isotype IgG1 control antibody (referred toin some instances as Cm7, Sigma M9269, at a final concentration of 1μg/mL) diluted in hybridoma maintaining media (HMM) were added to eachwell. Hybridoma maintaining media was made up of, Advanced DMEM (Gibco)supplemented with 1×Glutamax (Gibco), 20% v/v FBS (Gibco), 0.05 mMβ-Mercaptoethanol, 1×HT supplement (Gibco), and 1×penicillin/streptomycin (Gibco). Plates were incubated for 1 hour at 4°C. Culture media was aspirated and 50 μL of goat anti-mouse Alexa Fluor790 (Jackson ImmunoResearch, 115-655-071) at 1000 ng/mL supplementedwith 0.2 μM DRAQ5 (Biostatus) diluted in FACS Buffer (PBS+1% w/vBSA+0.1% v/v NaN₃) were added. Plates were again incubated for 1 hour at4° C. Supernatant was aspirated and 25 μL of 4% v/v paraformaldehydeadded and plates were incubated 15 minutes at room temperature. Plateswere washed twice with 100 μL PBS and then the wash buffer wascompletely removed. Fluorescence intensity was read by scanning platesusing an Odyssey Infrared Imaging System (LI-COR®). Anti-mouse binding(800 nm channel) was normalised to cell number (700 nm channel)according to LI-COR® recommended algorithm. Percent effect wascalculated as detailed below (Equation 1). Total binding was definedusing reference antibody at a final assay concentration of 1 μg/ml. Nonspecific binding was defined using mouse IgG1 isotype control (Sigma) ata final assay concentration of 1 μg/mL. Wells were defined as hits wherepercent effect was greater than or equal to 5%.

Equation 1: Calculation of Percentage Effect from Primary Screen(LI-COR) and HTRF

(Using 800% Resp Values (LI-COR) or 665/620 nm Ratio (See Equation 2)(HTRF)

${{Percent}\mspace{14mu} {effect}} = \frac{{{sample}\mspace{14mu} {well}} - {{non}\mspace{14mu} {specific}\mspace{14mu} {binding}}}{{{total}\mspace{14mu} {binding}} - {{non}\mspace{14mu} {specific}\mspace{14mu} {binding}}}$

Non-specific binding=values from wells containing isotype control mouseIgG1 or HMM or buffer

Total Binding (Binding HTRF and LICOR)=values from wells containingreference antibody Total binding (OX40L/OX40RFc assay)=OX40L and OX40RFc

Primary Screen: Binding to Recombinant Human OX40L:

In parallel to screening for binding to CHO-S expressed OX40L,supernatants collected from hybridoma wells were also tested to assessthe ability of secreted antibodies to bind to hOX40L expressed as arecombinant protein (produced in-house, see details in Example 1).Binding of secreted antibodies to recombinant hOX40L were identified byHTRF® (Homogeneous Time-Resolved Fluorescence, Cisbio) assay formatusing biotinylated hOX40L. 5 μL of hybridoma supernatant was transferredto a white 384 well low volume non binding surface polystyrene plate(Greiner). Then 5 μL of biotinylated hOX40L (working concentration 20nM) diluted in HTRF buffer (PBS (Sigma)+0.53 M KF (Sigma)+0.1% w/v BSA(Sigma) was added. 5 μL of combined detection reagents Streptavidin D2(Cisbio) diluted 1:100 in HTRF assay buffer for final dilution 1:400 andgoat anti-mouse IgG (Southern Biotech) labelled with europium cryptate(Cisbio) diluted 1:100 in HTRF assay buffer for final dilution 1:400were added. The concentration of goat anti-mouse IgG (Southern Biotech)labelled with europium cryptate was batch dependent and in some cases adilution of 1:1000 was performed to achieve a final assay concentrationof 1:4000. To adjust the total assay volume to 20 μL, 5 μL of HTRF assaybuffer was added to all wells. To define non-specific binding, additionof positive control antibody or hybridoma media was replaced with HTRFassay buffer or HMM. The plate was left to incubate in dark for 3 hoursprior to reading time resolved fluorescence at 620 nm and 665 nmemission wavelengths using an EnVision plate reader (Perkin Elmer). Moredetails of the HTRF® assay technology can be found in Mathis (1995)Clinical Chemistry 41(9), 1391-1397. Data were analysed by calculating665/620 ratio and percent effect for each sample according to Equation 2and Equation 1 respectively.

Equation 2: Calculation of 665/620 Ratio

665/620 ratio=(sample 665/620 nm value)×10000

For clones derived from KM040-1 and KM055-1 a selection criteria ofgreater than or equal to 20 percent effect was applied by the inventorsto define a well as a hit from recombinant hOX40L binding as describedin Table 1.

Primary Screen: Human OX40L/Human OX40R Fc Binding Assay:

In order to determine whether supernatants collected from hybridomawells inhibited the binding of OX40L to OX40RFc, secreted antibodieswere tested in an OX40L/OX40RFc binding HTRF assay. 5 μL of hybridomasupernatant was transferred to a white 384 well low volume non-bindingsurface polystyrene plate (Greiner). Biotinylated OX40L was diluted inHTRF assay buffer to a working concentration of 2.4 nM and 5 μL added.OX40RFc was then diluted to working concentration of 4.8 nM and 5 μLadded. Non-specific binding was defined by replacing OX40RFc with assaybuffer or HMM. Streptavidin cryptate (CISBIO) and anti-human Fc D2(CISBIO) were diluted in HTRF assay buffer to working concentration of1:100 and 5 nM respectively. Plates were covered, protected from lightand incubated at room temperature for 3 hrs prior to reading timeresolved fluorescence at 620 nm and 665 nm emission wavelengths using anEnVision plate reader (Perkin Elmer). Data were analysed by calculating665/620 ratio and percent effect for each sample according to Equation 2and Equation 5 respectively.

For clones derived from KM040-1 and KM055-1, a selection criteria ofless than or equal to 90 percent of the assay signal of OX40 receptor Fcbinding to OX40L was applied by the inventors to define a well as a hitas described in Table 1.

Secondary Screen: Binding to Cell Expressed and Recombinant Human OX40L

To determine whether wells selected using the primary screen selectioncriteria had the required characteristics set by the inventors, a numberof assays were performed. Hybridoma clones selected as hits from primaryscreening were cultured for 3 days and the supernatants collected fromhybridoma cells were tested to assess whether the secreted antibodiesthat bind to CHO-S expressed hOX40L, in some case bind to untransfectedCHO-S cells and whether they neutralise recombinant OX40R Fc binding toCHO-S hOX40L and ability to neutralise OX40R binding to recombinantbiotinylated hOX40L.

Binding to CHO-S Expressed hOX40L and Receptor Neutralisation:

CHO-S cells expressing hOX40L or untransfected CHO-S cells, diluted inFACS buffer (PBS+1% w/v BSA+0.1% w/v NaN₃) were distributed to a 96 wellV-bottom plate (Greiner) at a density of 1×10⁵ cells per well. Cellswere washed with 150 μL of PBS and centrifuged at 300×g for 3 min.Supernatant was aspirated and 150 μL of PBS added. This wash step wasrepeated.

25 μL of hybridoma supernatant or purified antibody from hybridomasupernatant diluted in FACS buffer was added to the washed cells andincubated for 10-15 minutes. Reference Antibody or mouse IgG1 controlantibody (Sigma) were diluted in FACS buffer to 20 μg/mL and 25 μL addedto cells. 25 μL of human OX40R Fc (in-house) diluted to 1000 ng/mL inFACS buffer were then added to wells. Cells were incubated at 4° C. for30 minutes.

Cells were washed twice with 150 μL of PBS centrifuging after each washstep and aspirating supernatant (centrifuged at 300×g for 3 minutes).

To detect antibody and receptor binding, 50 μL of Goat anti-human IgG-PE(Jackson ImmunoResearch) and APC anti-mouse IgG (Jackson ImmunoResearch)diluted 1 in 500 in FACS buffer was added to the cells. Cells wereincubated 30 minutes at 4° C. in the dark.

Cells were washed twice with 150 μL of PBS centrifuging after each washstep and aspirating supernatant (centrifuged at 300×g for 3 minutes).

To fix cells 100 μL 2% v/v paraformaldehyde was added and cellsincubated for 30 minutes at 4° C., cells were pelleted by centrifugation300×g and the plates and resuspended in 50 μL of FACS buffer. PE and APCsignal intensity (geomean) was measured by flow cytometry using a BDFACS Array instrument.

% of control binding was calculated using geomean fluorescence asdescribed in equation 1 where total binding was defined as referenceantibody at 10 μg/mL and non-specific binding as mouse IgG1 antibody at10 μg/mL. % receptor binding was calculated using Equation 3.

Equation 3: Percentage of Receptor Binding (FACS) Based on GeomeanFluorescence

${\% \mspace{14mu} {of}\mspace{14mu} {Receptor}\mspace{14mu} {binding}} = {\frac{{{sample}\mspace{14mu} {value}} - {{non}\mspace{14mu} {specific}\mspace{14mu} {binding}}}{{{total}\mspace{14mu} {binding}} - {{non}\mspace{14mu} {specific}\mspace{14mu} {binding}}} \times 100}$

Non-specific binding=No antibody, no receptor

Total binding=receptor (OX40R) only binding (no inhibitor)+isotypecontrol at 10 μg/mL

Secondary Screen—HTRF Ligand/Receptor Neutralisation

To determine whether antibodies identified from primary screenneutralise OX40L binding to OX40RFc an human OX40L/human OX40R Fcbinding assay was performed as described for primary screen.

Plates were left to incubate in dark for 3 hours prior to reading timeresolved fluorescence at 620 nm and 665 nm emission wavelengths using anEnVision plate reader (Perkin Elmer). More details of the HTRF assaytechnology can be found in Mathis (1995) Clinical Chemistry 41(9),1391-1397. Data were analysed by calculating delta F as described inEquation 4 and percentage of receptor for each sample according toEquation 5.

Equation 4: Calculation of % DeltaF

${\% \mspace{14mu} {delta}\mspace{14mu} F} = {\frac{\begin{matrix}{\left( {{sample}\mspace{14mu} 665\text{/}620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right) -} \\\left( {{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\text{/}620\mspace{14mu} {nm}\mspace{14mu} {ratio}\mspace{14mu} {value}} \right)\end{matrix}}{\left( {{non}\text{-}{specific}\mspace{14mu} {control}\mspace{14mu} 665\text{/}620\mspace{14mu} {nm}\mspace{14mu} {ratio}} \right)} \times 100}$

Equation 5: Percentage of Receptor Binding (HTRF)

Based on Calculation of % deltaF (Equation 4) or 665/620 Ratio (Equation2)

${\% \mspace{14mu} {of}\mspace{14mu} {Receptor}\mspace{14mu} {binding}} = {\frac{{{sample}\mspace{14mu} {value}} - {{non}\mspace{14mu} {specific}\mspace{14mu} {binding}}}{{{total}\mspace{14mu} {binding}} - {{non}\mspace{14mu} {specific}\mspace{14mu} {binding}}} \times 100}$

Non specific binding=HMM or buffer+OX40L (no receptor)

Total binding=receptor (OX40R) and OX40L (no inhibitor)

Hit Criteria Selection from Secondary Screening:

A panel of hits were selected based on binding and neutralisationassays. Hits in CHO-S OX40L binding assay were defined by the inventorsas significant binding to CHO-S OX40L cells and no binding to CHO-Scells by FACS. Hits were further defined as having the ability tosignificantly reduce OX40RFc binding to recombinant OX40L (HTRF) andsignificantly reduce OX40RFc binding to hOX40L expressed on CHO cells.Data is summarised in Table 1. Apparent affinity measurements by SPRwere also considered.

Example 3 Antibody Lead Characterization

Based on the screening selected wells were expanded and murine/humanchimeric antibodies purified using a standard Protein G based affinitychromatography purification (see method below). The antibodies weresubjected to various assays to assess their ability to block hOX40Lbinding to it receptor OX40R, as well as the ability of each antibody tobind to human as well as Rhesus monkey OX40L with high apparentaffinity. To decipher which antibodies were the best, selected cloneswere tested using OX40L/OX40RFc HTRF assay and OX40L induced IL2 releasefrom primary human T-cells.

TABLE 1 mAb Lead Summary HTRF Receptor Primary T-cell Apparent ApparentNeutralisation Assay Affinity Affinity FACS IC₅₀ nM IC₅₀ nM hOX40LRhsOX40L Antibody Binding (+/−SEM) (+/−SEM) (nM) (nM) 10A07 YES +++ +++CNROR CNROR (hybridoma) 10A07 ND +++ +++ CNROR CNROR (human) 1.2 nM 0.83nM (+/−0.17) (+/−1.2) 2D10 YES +++ ND CNROR CNROR (hybridoma) 2D10 ND+++ +++ CNROR CNROR (human) 0.75 nM 0.81 nM (+/−0.04) (+/−0.06) 9H04YES + ND 5.3 ND (hybridoma) 19H01 YES ++ ND 2.2 ND (hybridoma) CNROR =Cannot resolve off-rate IC₅₀ data represents arithmetic mean +/−standard error of mean (SEM) for three independent experiements ordonors.

Materials and Methods:

Purification of Antibodies from Hybridoma Supernatant:

Antibodies were purified using Protein G affinity chromatography.Antibodies were eluted from the Protein G media using IgG Elute reagent(Pierce) and the eluted antibodies were buffer swapped into PBS prior touse. Antibody purity was assessed by SDS-PAGE analysis and quantified byspectrophotometer reading at OD280 nm.

Binding of antibodies purified from hybridoma supernatant was carriedout as described herein.

HTRF Ligand/Receptor Neutralisation:

The following methods were carried out with a titration of inhibitor inorder to establish the clone potency as measured by IC₅₀ values in theassay. Antibody purified from hybridoma was titrated by diluting in HTRFassay buffer and 5 μL of this titration transferred to a white 384 welllow volume non-binding surface polystyrene plate (Greiner). BiotinylatedOX40L was diluted in HTRF assay buffer to a working concentration of 2.4nM and 5 μL added. OX40RFc was then diluted to working concentration of4.8 nM and 5 μL added. Non-specific binding was defined by replacingOX40RFc with assay buffer or HMM. Streptavidin cryptate (CISBIO) andanti-human Fc D2 (CISBIO) were diluted in HTRF assay buffer to workingconcentration of 1:100 and 5 nM respectively. Plates were covered,protected from light and incubated at room temperature for 3 hrs priorto reading time resolved fluorescence at 620 nm and 665 nm emissionwavelengths using an EnVision plate reader (Perkin Elmer). Data wereanalysed by calculating delta F as described in Equation 4 andpercentage of receptor for each sample according to Equation 5 or insome cases Equation 6. IC₅₀ values were determined using GraphPad Prismsoftware by curve fitting using a four-parameter logistic equation(Equation 7).

Equation 6: Percentage of Receptor Binding (HTRF)

Based on Calculation of % DeltaF (Equation 8)

${\% \mspace{14mu} {of}\mspace{14mu} {Receptor}\mspace{14mu} {binding}} = {\frac{{sample}\mspace{14mu} {value}}{{total}\mspace{14mu} {binding}} \times 100}$

Total binding=receptor (OX40R) and OX40L (no inhibitor)

Equation 7: Four Parameter Logistic Calculation

Y=Bottom+(Top-Bottom)/(1+10̂((Log IC50−X)*HillSlope))

X=logarithm of concentration.

Y=specific binding (equation 6)

Top and Bottom=Plateus in same units as Y (specific binding)

Log IC₅₀ in same units as X. Y starts at Bottom and goes to Top with asigmoid shape. Specific binding decreases as X increases.

Profiling of Fully Human Recombinant Anti-OX40L Antibodies in HTRFLigand/Receptor Neutralisation Assay

In order to determine whether recombinantly expressed fully humanpurified IgG inhibit human OX40L binding to OX40RFc the following methodwas carried out. Fully human purified IgG or other inhibitor were testedin order to establish the clone potency as measured by IC₅₀ values inthe assay. Antibodies recombinantly expressed and purified were titratedby diluting in HTRF assay buffer and 5 μL of this titration transferredto a white 384 well low volume non-binding surface polystyrene plate(Greiner). Biotinylated OX40L was diluted in HTRF assay buffer to aworking concentration of 2.4 nM and 5 μL added. OX40RFc directlylabelled with AF647 was then diluted to working concentration of 10 nMand 5 μL added. Non-specific binding was defined by replacingOX40RFc-AF647 with assay buffer or HMM. Streptavidin cryptate (CISBIO)was diluted in HTRF assay buffer to working concentration of 1:100 and 5μL added to all wells of the plate. Plates were covered, protected fromlight and incubated at room temperature for 3 hrs prior to reading timeresolved fluorescence at 620 nm and 665 nm emission wavelengths using anEnVision plate reader (Perkin Elmer). Data were analysed by calculatingdelta F as described in Equation 4 and percentage of receptor for eachsample according to Equation 5 or in some cases Equation 6. IC₅₀ valueswere determined using GraphPad Prism software by curve fitting using afour-parameter logistic equation (Equation 7) (FIG. 1).

Determining Effect of Anti-OX40L Antibodies on Recombinant OX40L InducedIL2 Release from Primary Isolated T-Cells

Recombinant human OX40L (in house) was diluted in culture media to aconcentration of 400 ng/mL and 50 μL added to a tissue culture treated96 well plate (Costar). Anti-OX40L antibodies or appropriate speciesisotype control (Sigma or in house) were titrated in culture media in a96 well plate (greiner) and then 50 μL of titration transferred to the96 well plate containing 50 μL OX40L. The antibody titration wasincubated for 30 minutes at room temperature with the recombinant OX40Lbefore CD3 positive T-cells were added.

PBMCs were isolated from leukoreduction system chambers (NHSBT) usingFicoll-Paque plus (GE Healthcare) by density gradient centrifugation.CD3 positive cells (T-cells) were isolated from human PBMC by negativeselection using magnetic microbeads (Miltenyi Biotech) according tomanufacturer's recommendations. The isolated cells were centrifuged at300×g/5 min, resuspended in culture media (culture media was defined aseither RPMI (Gibco)+10% v/v FBS or RPMI+5% v/v human AB serum) and 50 μLof the cell suspension added to the 96 well plate containing therecombinant OX40L and antibody titration to a achieve finalconcentration of 2×10⁵ cells/well.

Then 50 μL of PHA at 8 μg/mL was added to all wells to achieve a finalassay concentration of 2 μg/mL. The cells were incubated at 37° C. for 3days before supernatant were harvested and analysed for IL-2concentration. Maximal IL-2 release was defined by OX40L stimulation inthe absence of inhibitor. Minimal IL-2 release was defined by culturemedia only (no OX40L).

IL-2 levels in supernatants were determined using human IL-2 DuosetELISA kit (R & D) Systems) according to manufacturer's recommendations.IL-2 capture antibody (4 μg/mL diluted in PBS, 50 μL/well) was adsorbedto 96 well low auto-fluorescent, high protein binding plates (Costar)overnight at 4° C. Excess IgG was removed by washing with PBS-Tween andthe wells were blocked with 1% bovine serum albumin (BSA) in PBS for 1hour at room temperature, after which plates were washed as describedpreviously. 50 μL/well of conditioned culture media was then added IL-2standards (from 2000 pg/mL, 1:2 dilution) were also added to ELISAplates as an ELISA control and the plates were incubated at roomtemperature for at least 1 hour.

Following incubation, plates were washed as before to remove unboundproteins. Biotinylated IL-2 detection Ab (200 ng/mL in reagent diluent(0.1% BSA/PBS); 50 μL/well) was then added to the plates and incubatedat RT for 1 h. Unbound detection antibody was removed by washing withPBS-Tween (0.1% v/v), while the remaining biotinylated antibody wasdetected by streptavidin-Europium3+conjugate (DELFIA® detection,PerkinElmer). Time-resolved fluorescence was measured at 615 nm on anEnvision plate reader (PerkinElmer). Fluorescence data was plotted asEuropium counts or concentration of IL-2 release calculated fromstandard curve by linear regression according to manufacturer'srecommendations. IC₅₀ values were determined using GraphPad Prismsoftware by curve fitting using a four-parameter logistic equation(Equation 7).

Surface Plasmon Resonance Analysis:

SPR analysis was carried out using the ProteOn™ XPR36 Array System(BioRad). Anti-mouse IgG (GE Healthcare BR-1008-38) was immobilised on aGLM biosensor surface using amine coupling, the surface was then blockedusing 1 M ethanolamine. Test antibodies were captured on this surfaceand recombinant hOX40L (human and rhesus) were used at a singleconcentration of 256 nM, binding sensorgrams were double referencedusing a buffer injection (i.e. 0 nM) to remove baseline drift andinjection artefacts. Apparent affinities for the OX40L-antibodyinteraction were determined using the 1:1 model inherent to the ProteOnXPR36 analysis software. The assay was run using HBS-EP (Teknova) asrunning buffer and carried out at 25° C.

Example 4 Sequence Recovery of Lead Antibody Candidates

After the selection and characterization of lead candidates, their fullyhuman variable domains were recovered using RT-PCR using a mixture offorward and reverse primers. Antibodies were reformatted into a humanIgG4 backbone (IgG4-PE) and expressed using a transient expressionsystem in CHO-S cells. A summary of all sequences is displayed in theSequence Listing.

RNA Isolation from Hybridoma Cells:

Total RNA was extracted from hybridoma cells using TRIzol™ Reagent(Invitrogen). The quantity and quality of the isolated RNA was analysedspectrophotometrically.

Antibody Variable Domain Recovery by RT-PCR:

Selected clones were used for preparing total RNA, which was used in anRT-PCR reaction to recover the heavy chain V-regions. IgG specificreverse primers and Ig leader sequence specific forward primer sets oralternatively IgG specific reverse primers and Ig 5′ untranslated region(UTR) sequence specific forward primer sets were used for the heavychains. Kappa constant region specific reverse primers and kappa leadersequence specific forward primer sets or alternatively Kappa constantregion specific reverse primers and kappa 5′UTR sequence specificforward primer sets were used for the kappa OX40L chains. The RT-PCRproducts were separated by agarose gel electrophoresis with the DNA ofthe predicted size being sequenced in the forward and reversedirections. Alternatively, the RT-PCR products were subcloned into acloning vector and DNA of individual colonies submitted for sequencing.

Cloning of Recombinant Antibodies

DNA encoding the heavy chain variable region of mAb 10A7 was cloned intoa pREP4 expression plasmid (Invitrogen) in frame with the Human IgG1constant region and DNA encoding the light chain variable region of mAb10A7 was cloned into a pREP4 expression plasmid in frame with the HumanKappa constant region using standard restriction enzyme digestion andligation.

The heavy chain variable region coding sequences of mAbs 10A7 and 2D10in frame with the Human IgG4-PE constant region were codon optimized formammalian expression and cloned into a pXC-18.4 expression plasmid(Lonza) and the light chain coding sequences of mAbs 10A7 and 2D10 inframe with the Human Kappa constant region were codon optimized formammalian expression and cloned into a pXC-17.4 expression plasmid(Lonza) using standard restriction enzyme digestion and ligation. Forthe simultaneous expression of the heavy and light chains the vectors apXC-17.4 and a pXC-18.4 were fused into one single vector using standardrestriction enzyme digestion and ligation.

All constructs were sequenced to ensure their correct sequencecomposition.

Transient Expression of OX40L Antibodies

Antibodies were expressed transiently to produce recombinant proteinusing Invitrogen's FreeStyle™ CHO-S suspension adapted cell line.Plasmids were transfected into the cells using PEI (polyethylenimine1\4W 40000) and left to overgrow for a period of 13 days beforeharvesting the supernatant for purification. Cells were fed during theovergrow process with ActiCHO™ Feeds A and B from GE Healthcare to helpboost productivity and promote longevity of the cells. During theovergrow process samples were taken regularly to monitor cell growth andviability.

Generation of Stable Lonza Pools

In order to produce the gram amounts required for toxicology studies,10A7 and 2D10 OX40L antibodies were transferred to the Lonza GS Xceedsystem for stable expression. The HC and LC for each antibody was firstcodon optimised for expression in CHO cells by Genewiz. The HC cassette(containing the optimised IgG4PE constant region) was then cloned intoLonza's pXC18.4 vector and LC cassette (containing the optimised kappaconstant region) cloned into Lonza's pXC17.4 vector using standardrestriction enzyme digestion and ligation. A double gene vector (DGV)encoding both the HC and LC sequences was then created by restrictionenzyme digestion and ligation and sequence confirmed before expression.

Prior to stable pool creation; the single gene vectors encoding the HCand LC's separately as well as the DGV containing both, were expressedin the Lonza CHOK1SVKO cell line transiently using PEI (polyethylenimineMW 40000). Cells were left to overgrow for a period of 13 days beforeharvesting the supernatant for purification. During this period cellswere fed with ActiCHO™ Feeds A and B from GE Healthcare to help boostproductivity and promote longevity of the cells. During the overgrowprocess samples were taken regularly to monitor cell growth andviability. Once transient expression was confirmed and purified materialanalysed the antibodies were expressed as stable pools.

Stable pools were generated using Lonza's proprietary methods and media.4 pools were created per antibody and left to recover over a period of10-15 days. After the cells had recovered, pre-seed stocks (PSS) ofcells were frozen down for later recovery and creation of MCB. Smallscale (50 mL) shake flask fed batch overgrows were then set up usingLonza's proprietary media. Cells were left to overgrow for a period of14 days. During this period cells were monitored for growth, viabilityand glucose levels. Cells were supplemented accordingly with Lonza'sproprietary feed and 400 g/L glucose. Samples were also taken throughoutthe process for crude sample quantification. At the end of the overgrowprocess the supernatant was harvested for purification.

Stable pools were generated using Lonza's proprietary methods and media.4 pools were created per antibody and left to recover over a period of10-15 days. After the cells had recovered, pre-seed stocks (PSS) ofcells were frozen down for later recovery and creation of MCB. Smallscale (50 mL) shake flask fed batch overgrows were then set up usingLonza's proprietary media. Cells were left to overgrow for a period of14 days. During this period cells were monitored for growth, viabilityand glucose levels. Cells were supplemented accordingly with Lonza'sproprietary feed and 400 g/L glucose. Samples were also taken throughoutthe process for crude sample quantification. At the end of the overgrowprocess the supernatant was harvested for purification.

Whilst the 2D10 and 10A07 were similar in sequence, there expressionprofiles in the stable Lonza pools were different, 10A07 expressed tovery low titres, whereas 2D10 expressed at much greater titres (seeTable 2) under optimal conditions when using shake flasks in 4 separategenerated stable pools.

TABLE 2 (Concentration in mg/L) Day Day Day Day Day Day Day Day Stablepool 7 8 9 10 11 12 13 14 2D10-1 261 492 681 993 1157 1590 1530 15752D10-2 245 461 665 983 1127 1485 2025 1995 2D10-3 317 528 731 1163 13671785 1905 1860 2D10-4 372 677 785 1286 1350 1935 1965 1800 Control 92129 167 229 297 357 416 N/A Antibody 1 Control 66 95 127 161 208 238 266N/A Antibody 1 Control 68 102 132 192 266 324 314 N/A Antibody 1 Control88 129 165 245 328 410 385 N/A Antibody 1

After expression, the antibody to be used in the Rhesus Macaque GvHDmodel was purified using a two-step purification process. The antibodieswere first purified using MabSelect SuRe (GE Healthcare) affinitychromatography. Antibodies were eluted from the MabSelect SuRe mediausing IgG Elute reagent (Pierce) and the eluted antibodies were dialysedin sodium acetate (pH 5.5) buffer prior to the second purification step.Antibodies were then purified by cation exchange and eluted with sodiumchloride in sodium acetate buffer. Eluted antibodies were dialysed inPBS. Antibodies were quantified by spectrophotometer reading at OD280 nmand adjusted to the desired concentration (10 mg/ml). Antibody puritywas assessed by SDS-PAGE analysis and size exclusion chromatography.Endotoxin concentration was measured with Endosafe PTS and LAL TestCartridges (Charles River Laboratories).

Example 5 Determining Effect of Anti-OX40L Antibodies in Allogeneic PBMCMixed Lymphocyte Reaction

PBMCs are isolated from leukoreduction system chambers (NHSBT) usingFicoll-Paque plus (GE Healthcare) density gradient centrifugation. PBMCare pre-incubated with mitomycin C (Sigma) at 10 μg/mL in PBS for onehour at 37° C. Cells are then washed 3 times in PBS centrifuging at300×g for 3 minutes, aspirating the supernatant after each wash.Allogeneic PBMC (not treated with mitomycin C) are added to a 96-wellplate in RPMI supplemented with 10% v/v FBS at a concentration of2×10⁶/ml, 50 μL/well. Anti-OX40L antibodies are diluted in culture mediaand added to 96 well plate containing PBMC (not mitomycin C treated) at50 μL/well. Mitomycin C treated PBMC are then added to allogeneic PBMC(not treated with mitomycin C) in 96-well plate at a final cell ratio inrange of 1:1 to 4:1 mitomycin C treated to non mitomycin C based onnumber of cells/well. The cells are incubated for five days at 37° C./5%CO₂. After five days TNF-α, IFN-γ, and IL-2 are measured by duoset ELISA(R&D Systems) according to manufacturer's recommendations. Proliferationis measured by CFSE dilution according to manufacturer'srecommendations.

PBMCs were isolated from leukoreduction system chambers (NHSBT) usingFicoll-Paque plus (GE Healthcare) density gradient centrifugation. PBMCwere pre-incubated with mitomycin C (Sigma) at 10 μg/mL in PBS for onehour at 37° C. Cells were then washed 3 times in PBS centrifuging at300×g for 3 minutes, aspirating the supernatant after each wash.T-lymphocytes (T-cells) in some cases CD3 positive and in other casesCD4 and CD8 positive were isolated from allogeneic PBMC by negativeselection using magnetic microbeads (Miltenyi Biotech) according tomanufacturer's recommendations. In some cases, non-mytomycin C treatedPBMC were used instead of T-cells. The isolated cells were centrifugedat 300×g/5 min, resuspended in culture media (culture media was definedas either RPMI (Gibco)+10% v/v FBS or RPMI+5% v/v human AB serum) and 50μL of the cell suspension added to the 96 well plate containing therecombinant OX40L and antibody titration to a achieve finalconcentration of 2×10⁵ cells/well. Anti-OX40L antibodies were diluted inculture media to a final assay concentration 100 nM or in some cases atitration of antibody was used. The antibodies were added to 96 wellplate containing T-cells or non-mytomycin C treated PBMC at 50 μL/well.Mitomycin C treated PBMC were then added to Tcells or non mytomycin Ctreated PBMC in 96-well plate at a final cell ratio in range of 1:1 to4:1 mitomycin C treated PBMC to T-cells (or PBMC) based on number ofcells/well. The cells were incubated for five days at 37° C./5% CO₂.After five days, IFN-γ was measured by duoset ELISA (R&D Systems)according to manufacturer's recommendations.

Anti-OX40L antibodies were defined as inhibitors in allogeneic PBMC/Tcell MLR or PBMC/PBMCl MLR when >20% inhibition (see Equation 8) offactor release (IFN-γ,) or were observed relative to control wells inthe absence of antibody. From four experiments performed, one experimentwas a technical failure, defined as no MLR response (IFN-γ release)detected between allogeneic donors. Of the three remaining experiments,all three showed inhibition (>20% inhibition of factor release (IFN-γ,)observed relative to control wells in the absence of antibody) with2D10, 10A07 and positive control 1, however in one of three experiments,significant inhibition was also observed with the isotype controlantibody (FIG. 2). For PBMC/PBMC MLR, three experiments were performed.Of three experiments, two were regarded as technical failure as therewas no or low IFN-γ release. However, in another experiment 10A07inhibited IFN-γ release when compared to the isotype control.

Equation 8: Percentage Inhibition (MLR)

Based on Values from IFN-γ or IL2 Release (pg/mL) Determined asDescribed

${\% \mspace{14mu} {inhibition}} = {100 - {\frac{{{sample}\mspace{14mu} {value}} - {{no}\mspace{14mu} {stimulus}}}{{{No}\mspace{14mu} {IgG}} - {{no}\mspace{14mu} {stimulus}}} \times 100}}$

No Stimulus=wells where only T-cells or non-mytomycin C treated PBMC areadded (no mitomycin C treated PBMC)

No IgG=wells where T-cells or in some cases non-mytomycin C treated PBMCalong with mytomycin C treated PBMC are added but no IgG

Example 6 Determining Effect of Anti-OX40L Antibodies on CD3 PrimedPrimary Human T Lymphocytes

In order to determine whether anti-OX40L had the ability to induceT-cell responses in the absence of OX40L, the assay below was performedusing method adapted from Wang et al., Hybridoma (Larchmt)., 2009August; 28(4):269-76, in which an agonist anti-OX40L antibody wasdescribed.

A mouse anti-human CD3 antibody (Becton Dickinson) was diluted to 0.5μg/mL in sterile PBS and 50 μL/well added to a 96 well high bindingsterile plate and incubated overnight at 4° C.

Following overnight incubation, the plate was washed three times with100 μL of sterile PBS.

T-cells (CD3 positive) were isolated from PBMC derived fromleukoreduction system chambers (NHSBT) as described in Example 3.Following isolation, the cells were added to wells in 100 μL to achievea final concentration of 1×10⁵ cells/well.

Test antibodies were diluted in RPMI+10% FBS and 50 μL or 100 μL/welladded to cell plate to achieve a final assay concentration of 10 μg/mL.In some cases, a mouse anti-human CD28 antibody (Becton Dickinson) wasalso added to wells at a final concentration of 1 μg/ml

The assay was incubated for 5 days. After 5 days, harvest supernatantsand IFN-γ levels in supernatant were determined as described in Example5.

The assay was performed in four independent donors and no effect ofadding 10A07 or 2D10 in IgG4PE format was observed (IFN-γ release) overthat observed with human IgG4PE isotype control.

Example 7 Rhesus Macaque Graft Versus Host Disease (GvHD) Model

The effectiveness of antibody 2D10 IgG4PE as a monotherapy prophylacticfor the prevention of GvHD was examined in a Rhesus Macaque model ofhaploidentical hematopoietic stem cell transplantation (HSCT). It hadbeen previously described that monkeys undergoing HSCT in this model hada survival time of 6-8 days (Miller, Weston P., et al. “GVHD afterhaploidentical transplantation: a novel, MHC-defined rhesus macaquemodel identifies CD28⁻ CD8⁺ Tcells as a reservoir of breakthrough T cellproliferation during costimulation blockade and sirolimus-basedimmunosuppression.” Blood, 116, 24(2010):5403-5418.)

All transplants were between half-sibling pairs that are mismatched atone MHC haplotype (“haploidentical-HCTs”). Recipient animals hadirradiation based pre-myeloablative pre-transplant conditioning using alinear accelerator. Dose rate: 7 cGy/min. Dose 1020 cGy given in 4fractions. The leukopheresis donor animal underwent GCSF mobilisationand underwent leukopheresis using a Spectra Optia apheresis machine. Thetable below gives the dose per kg of total nucleated cells (TNC) dose ofCD3⁺ cells, and CD34⁺ cells for the four successful experiments.

TABLE 3 Recipient Recipient Animal Bodyweight TNC CD3⁺ T-cells CD34⁺cells ID# No. (kg) (10⁹/kg) 10⁶/kg 10⁶/kg A14079 #2 9.75 1.13 149.760.51 A14081 #4 7.02 2.99 389.08 4.79 A14082 #5 7.6 2.24 312.95 2.69A14087 #6 5.75 3.44 385.66 9.99

2D10 IgG4PE was dosed at 10 mg/kg i.v. according to a planned dosingschedule to take place on Day −2, Day +5, Day +12, Day +19, Day +26, Day+33, Day +40, Day +47 post-transplant. No serious adverse dosing sideeffects were seen with any of the animals as a result of administering2D10 IgG4PE.

Samples were taken during the course of the study to monitor donorchimerism (Table 4) and white blood cell counts. The primary end pointwas based on survival, with a survival to 15 days deemed to be a sign ofsuccessful prophylactic therapy (and compared to the documented survivalof 6-8 days with no prophylaxis; Miller et al 2010, supra). Though fullpathology and histology with GvHD grading scores, markers of T-cellproliferation and activation (such as Ki-67 and granzyme B) and genearray analysis are planned, they were not available for inclusion at thetime of drafting.

Methods for these studies are essentially as described in Miller W P etal., (2010) “GVHD after haploidentical transplantation: a novel,MHC-defined rhesus macaque model identifies CD28−CD8+ T cells as areservoir of breakthrough T-cell proliferation during costimulationblockade and sirolimus-based immunosuppression”, Blood 116:5403-5418.

Clinical Staging of GvHD

Scoring of clinical symptoms was based on observational assessments andclinical chemistry, classified according to the criteria set out inTable 5.

Histopathology

Tissues, including lung, liver, skin and gastrointestinal tract werecollected at necropsy and fixed in formalin and paraffin-embedded.Sections were cut, slide-mounted and stained with haematoxylin/eosin orwith T cell markers for visualisation of tissue infiltration bylymphocytes. Prepared slides are read by a histopathologist withspecific expertise in GvHD using a semiquantitative scoring system.

Flow Cytometry

Longitudinal peripheral blood samples were collected before and afterhaematopoietic stem cell transplant and at necropsy for flow cytometricanalysis of lymphocyte subsets. Lung, liver, colon spleen and lymph node(axillary and inguinal) tissues were collected at necropsy anddissociated or enzymatically digested as appropriate for subsequentanalysis of lymphocyte infiltrates by flow cytometry. Samples wereanalysed by multicolour flow cytometry using a LSR Fortessa cellanalyser (BD Biosciences) using the following T lymphocyte markerprobes: CD3 (APC-Cy7 label; clone SP34-2, BD Biosciences), CD4 (BV786label; clone L200, BD Biosciences), CD8 (BUV395 label; clone RPA-T8, BDBioscences), CD28 (PE-Cy7 label; clone CD28.2, eBioscience), CD95 (BV605label; clone DX2, Biolegend). Proliferating cell populations wereidentified using Ki-67 (FITC label, Dako). CD4+ or CD8+ T cellsubcompartments were labelled as follows: naïve T-cells (CD28+/CD95−),central memory T-cells (CD28+/CD95+), effector memory T-cells(CD28−/CD95+).

Blood was collected into tubes with Sodium EDTA, and then red bloodcells were lysed with lysis buffer containing ammonium chloride.Remaining leukocytes were washed with FACS buffer (PBS with 2% FBS) andstained with antibody cocktail (Table 7) for 30 minutes at 4° C. Afterstaining, cells were washed and fixed in 1×BD Stabilizing Fixative.Acquisition of flow data was performed on BD LSR Fortessa cytometer.Data were analyzed using FloJo. T-cells were defined asCD3+CD14/CD20-lymphocytes.

TABLE 7 List of used antibodies for T cell immunophenotyping by flowcytomerty Antibody Fluorochrome Clone Company CD3 APC-Cy7 SP34-2 BDBiosciences CD4 BV786 L200 BD Biosciences CD8 BUV395 RPA-T8 BDBiosciences CD14 PerCP-Cy5.5 M5E2 BD Biosciences CD20 PerCP-Cy5.5 2H7eBioscience CD28 PE-Cy7 CD28.2 eBioscience CD45RA APC 2H4LDH11LDB9Beckman Coulter CD95 BV605 DX2 Biolegend CCR7 (CD197) BV421 G043H7Biolegend OX40 (CD134) PE L106 BD Biosciences

Results:

1: Expansion of Memory Stem T-Cells after Transplantation

In a non-human primate model of acute Graft-versus-Host disease (GVHD),allogeneic hematopoietic cell transplantation (HCT) results in earlyexpansion of both CD4 and CD8 memory stem T-cells (Tscm:CD45RA+CCR7+CD95+) at the expense of reconstitution of bona fide naïveT-cells (Tn: CD45RA+CCR7+CD95−) (FIG. 3). These Tscm cells circulate inthe blood, and also reside in both lymphoid (lymph nodes, spleen) andnon-lymphoid organs (lung, liver and colon).

2: 2D10 IgG4PE Limits Expansion of Tscm

Treatment with the blocking anti-OX40L antibody, 2D10 IgG4PE, results inprolonged survival of animals after allogeneic HCT and reduces clinicalsymptoms of acute GVHD. This delay in GVHD progression was associatedwith limited CD4+ Tscm expansion and preservation of CD4+ Tn cells (FIG.4).

3: CD4 Tscm Cells Express OX40 on their Surface

As shown in FIG. 5, CD4+ Tscm express OX40 on their surface, but naïveT-cells do not. Moreover, the level of OX40 expression was comparablebetween CD4+ Tscm and central memory cells (Tcm). Importantly, OX40expression was detected on CD4+ Tscm cells broadly. They are detected innaïve monkeys before transplantation (both in the blood and lymphoidorgans), as well as in leukopheresis products. This expression is alsoseen in allogeneic HCT recipients longitudinally after transplantation.

4: Comparative Analysis of Tscm in 02D10 Ig4PE Treated Animals Comparedto Standard GvHD Therapies

The proportion of post-HCT Tscm cells evident in the peripheral blood ofrhesus monkeys that received 02D10 IgG4PE were compared with Tscm fromseparate groups of animals administered either sirolimus (rapamycin) ora combination of tacrolimus plus methotrexate (Tac/MTX). The results forCD4+ Tscm cells are shown in FIG. 6a , and the results for CD8+ Tscmcells are shown in FIG. 6b . Data indicate that treatment withanti-OX40L antibody 02D10 IgG4PE results in a sustained inhibition ofthe proportion of Tscm cells compared with the sirolimus and Tac/MTXtreatment.

Conclusions:

An OX40-expressing subset of Tscm might be sensitive to 2D10IgG4PE-mediated OX40L-blockade. This blockade may control Tscm expansionand therefore limit the progression of acute GVHD. The OX40 pathway is apotentially novel mechanism of Tscm regulation, which can be used inclinical practice to treat immune-mediated diseases or improve theoutcome of adoptive immunotherapy.

Chimerism

Peripheral blood or T cell (CD3+/CD20−) chimerism was determined usingdivergent donor- and recipient-specific MHC-linked microsatellitemarkers, by comparing peak heights of the donor- and recipient-specificamplicons (Penedo M C et al., (2005) “Microsatellite typing of therhesus macaque MHC region”, Immunogenetics 57:198-209).

TABLE 5 Stage Skin Liver (Billirubin) GI 0 No GVHD rash <4-fold increaseNo over baseline diarrhea 1 Rash <25% of 4- to-8-fold increase “Mild”surface area diarrhea 2 Rash 25-50% of 8- to 20-fold increase “Moderate”surface area diarrhea 3 Rash >50% of 20- to 50-fold increase “Severe”surface area diarrhea 4 Generalized >50-fold increase “Very severe”erythroderma diarrhea with bullous formation

A total of six animals were selected to receive HSCT. Of these 6animals, two of the experiments were deemed a technical failure, oneanimal experienced viral reactivation which may have hamperedengraftment and it was seen that donor chimerism initially climbed butthen dropped, indicating that second reconstitution was autologousrepopulation. A single high cytomegalovirus (CMV) and Rhesus macaqueLymphocryptovirus (rhLCV) reading was seen at the same time as the dropin chimerism and autologous repopulation. The second technical failurewas the result of failure of the apheresis machine to produce a suitableproduct for transplantation. Since the recipient animal had already beenirradiated, it had to be sacrificed. The four other animals all survivedto the primary endpoint of 15 days, exhibiting extended survivalcompared to both historical and contemporaneous no-prophylaxis controls.Table 6 below outlines the summary of each animal in this study.

Example 8 Pharmacokinetics

Rhesus macaques were dosed with 10 mg/kg of 2D10 or appropriatenon-functional isotype control antibody on Day 0. Samples were taken,after +15 minutes, +1 hour, +8 hours, +24-36 hours, +72 hours, +96hours, +Day 8, +Day 11, +Day 15, +Day 18, +Day 22, +Day 25. On Day 29,animals were dosed with 3 mg/kg of 2D10 or appropriate non-functionalisotype control antibody. Samples were taken on Day 29 after +15minutes, +1 hour, +8 hours and then 24-36 hours after Day 29. Samplescontinued to be taken on +Day 32, +Day 33, +Day 36, +Day 39, +Day 43,+Day 46, +Day 50, +Day 53, +Day 57, +Day 60, +Day 64, +Day 67 and +Day71.

To determine the PK, anti-human IgG is diluted to 8 μg/mL in PBS and isadsorbed to 96 well low auto-fluorescent, high protein binding plates(Costar) overnight at 4° C. Excess IgG is removed by washing withPBS-Tween and wells are blocked with 5% w/v non-fat dried milk (blockingbuffer) for 1 hour at room temperature. Following incubation period,plates are washed. Plasma samples are diluted in blocking buffer(multiple dilutions). A standard curve is also generated using atitration of positive control anti-OX40L antibody diluted in blockingbuffer from 10 μg/mL (1 in 3 dilution). Either titration or dilutedplasma sample are added to plate and incubated for 1 hr at roomtemperature. Plates are then washed and biotinylated human OX40L isdiluted to 500 ng/mL in blocking buffer added for 1 hour at roomtemperature. Plates are then washed and streptavidin-Europium3+conjugate(DELFIA® detection, PerkinElmer) diluted in DELFIA® assay buffer (PerkinElmer) is added. Plates are then washed 3 times in Tris BufferedSaline+0.1% tween. Then, DELFIA Enhancement solution (Perkin Elmer) isadded to the plate and time-resolved fluorescence is measured at 615 nmon an Envision plate reader (PerkinElmer). The concentration ofanti-OX40L antibody in the plasma is calculated by extrapolatingfluorescence values from sample wells to those obtained from thestandard curve generated from the titration of the positive controlanti-OX40L antibody using a four parameter logistics curve fittingalgorithm.

Example 9

Rhesus Macaque (GvHD) Model: Effect of Combined Prophylaxis with 2D10IgG4PE Plus Rapamycin

A further rhesus macaque GvHD study was conducted to determine theeffect of combined post-HSCT prophylaxis with 2D10 IgG4PE and rapamycin.The study was performed as described in Example 7, with dosing asfollows: 2D10 IgG4PE was administered i.v. at 10 mg/kg on Day −2, Day+5, Day +12, Day +19, Day +26, Day +33, Day +40, Day +47 and Day +56post-transplant. Rapamycin was administered at a loading dose of 0.1mg/kg i.m. on Day −14, followed by daily i.m. maintenance doses of 0.025mg/kg until the scheduled termination of the study at Day +100.Rapamycin dosing was adjusted to maintain serum trough levels within therange 5-15 ng/mL.

Results:

Post-HSCT administration of 2D10 IgG4PE together with rapamycin resultedin extended GvHD-free and absolute survival (median survival time,MST >82 days; n=3) compared to historical control animals that did notreceive post-HSCT experimental treatment (MST=8 days; n=4; Furlan et al,Science Translational Medicine, Vol 7 (315); 315ra1910). The effect ofcombined 2D10 IgG4PE plus rapamycin dosing appeared also to be greaterthan the additive effect of each molecule when administered alone (FIG.7: MST for post-HSCT 2D10 IgG4PE and rapamycin were 19 and 17 days,respectively; both n-4). It is also noted that Furlan et al discloses aMST for combined prophylaxis with tacrolimus plus methotrexate of 49days. It is expected that a combination of tacrolimus plus methotrexateand an anti-OX40L antibody (such as 2D10), or indeed a combination oftacrolimus and an anti-OX40L antibody (such as 2D10), would also providethe synergistic results as seen in this Example.

TABLE 4 Ani- Ani- Survival Whole Blood Chimerism (%) mal mal DurationDay Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day Day No.ID (days) 0 1 4 5 6 7 8 11 12 14 15 16 18 20 21 23 26 (#1) (13189) (24)0 6.6 27.5 90.4 81.8 19.7 0 #2 14079 16 0 5.7 31.7 66.3 82.3 88.2 79.8(#3) (14075) (0) #4 14081 26 22.9 68.2 82.9 92.1 97.5 98.4 98.8 98.7 #514082 22 91.4 98.4 98.6 99.1 99.2 98.6 #6 14087 16 16.6 66.3 97.4 99.599.4 Data in brackets indicates experimental failure due to infection(animal 1) or technical failure (animal 3).

TABLE 6 2D10 IgG4PE Rhesus GvHD Study Animal Details #1 Survival to day24. Received 4 doses of 2D10 IgG4PE. Biphasic hematopoieticreconstitution; peripheral blood chimerism data indicated initial donorengraftment followed by autologous repopulation concurrent with evidenceof CMV and rhLCV infection. Viral infection considered possible cause ofgraft failure. Recorded as Technical Failure. #2 Survival to Day 16.Received 3 doses of 2D10 IgG4PE. Peak peripheral blood donor chimerismof 88% at Day 15. No evidence of CMV or rhLCV infection. Studyterminated on veterinary advice due to wound at catheter site (notdeemed to be treatment or GvHD related). GvHD staging at necropsy: skin1 (rash <25%); liver 0 (no bilirubin elevation); GI 0 (no diarrhoea). #3Recorded as Technical Failure. Apheresis equipment failure resulted indrastically suboptimal donor blood product. #4 Survival to Day 26.Received 4 doses of 2D10 IgG4PE. Clear hematopoietic reconstitution withpeak peripheral blood donor chimerism of 99% by Day 23. No evidence ofCMV or rhLCV infection. Study terminated on veterinary advice due toscrotal oedema. GvHD staging at necropsy: skin 2 (rash 25-50%); liver 0(no bilirubin elevation); GI 0 (no diarrhoea). Gross necropsy confirmedno overt visceral GvHD. #5 Survival to Day 22. Received 4 doses of 2D10IgG4PE. Clear hematopoietic reconstitution with peak peripheral blooddonor chimerism of 99% by Day 12. No evidence of CMV or rhLCV infection.Study terminated due to persistent low platelet count with high bleedingrisk and developing signs of acute systemic GVHD. GvHD staging atnecropsy: skin 3 (rash >50%); liver 1 (4-8 x bilirubin elevation); GI 3(severe diarrhoea). #6 Survival to Day 16. Received 3 doses of 2D10IgG4PE. Clear hematopoietic reconstitution with peak peripheral blooddonor chimerism of 100% on Day 12. No evidence of CMV or rhLCVinfection. GvHD staging at necropsy: skin 2 (rash 25-50%); liver 1 (4-8x bilirubin elevation); GI 2 (moderate diarrhoea).

SEQ ID NO: 1 10A07 VH Nucleotide SequenceGAGGTGCAACTGGTGGAGTCTGGGGGAGTCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATATTATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGTAGTGGTGGTGGTACATACTACGCAGACTCCATGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCCGTATATTACTGTGCGAAAGATCGGTTAGGTCCGATTACTTTGGTTCGGGGGGGCTATTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGG TCACCGTCTCCTCA 2 VH AminoAcid Sequence EVQLVESGGVLVQPGGSLRLSCAASGFTFSSYIMTWVRQAPGKGLEWVSGISGSGGGTYYADSMKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCAKDRLGPITLVRGGYYYGMDVWGQGTTVTVSS 3 HCDR1 Nucleotide Sequence (IMGT)GGATTCACCTTTAGCAGTTATATT 4 HCDR1 Amino Acid Sequence (IMGT) GFTFSSYI 5HCDR2 Nucleotide Sequence (IMGT) ATTAGTGGTAGTGGTGGTGGTACA 6 HCDR2 AminoAcid Sequence (IMGT) ISGSGGGT 7 HCDR3 Nucleotide Sequence (IMGT)GCGAAAGATCGGTTAGGTCCGATTACTTTGGTTCGGGGGGGCTATT ACTACGGTATGGACGTC 8 HCDR3Amino Acid Sequence (IMGT) AKDRLGPITLVRGGYYYGMDV 9 HCDR1 NucleotideSequence (KABAT) AGTTATATTATGACT 10 HCDR1 Amino Acid Sequence (KABAT)SYIMT 11 HCDR2 Nucleotide Sequence (KABAT)GGTATTAGTGGTAGTGGTGGTGGTACATACTACGCAGACTCCATGA AGGGC 12 HCDR2 Amino AcidSequence (KABAT) GISGSGGGTYYADSMKG 13 HCDR3 Nucleotide Sequence (KABAT)GATCGGTTAGGTCCGATTACTTTGGTTCGGGGGGGCTATTACTACGG TATGGACGTC 14 HCDR3Amino Acid Sequence (KABAT) DRLGPITLVRGGYYYGMDV 15 VL NucleotideSequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCGACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGTTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGAGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCGTCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGACGTTCGGCCAAGGGACCAGGGTGGAAATCAAA 16 VL Amino Acid SequenceDIQMTQSPSSLSASVGDRVTITCRASQSISDYLNWYQQKPGKAPKFLIYAASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQSYSTPRTFG QGTRVEIK 17 LCDR1Nucleotide Sequence (IMGT) CAGAGCATTAGCGACTAT 18 LCDR1 Amino AcidSequence (IMGT) QSISDY 19 LCDR2 Nucleotide Sequence (IMGT) GCTGCATCC 20LCDR2 Amino Acid Sequence (IMGT) AAS 21 LCDR3 Nucleotide Sequence (IMGT)CAACAGAGTTACAGTACCCCTCGGACG 22 LCDR3 Amino Acid Sequence (IMGT)QQSYSTPRT 23 LCDR1 Nucleotide Sequence (KABAT)CGGGCAAGTCAGAGCATTAGCGACTATTTAAAT 24 LCDR1 Amino Acid Sequence (KABAT)RASQSISDYLN 25 LCDR2 Nucleotide Sequence (KABAT) GCTGCATCCAGTTTGCAAAGT26 LCDR2 Amino Acid Sequence (KABAT) AASSLQS 27 LCDR3 NucleotideSequence (KABAT) CAACAGAGTTACAGTACCCCTCGGACG 28 LCDR3 Amino AcidSequence (KABAT) QQSYSTPRT 29 Heavy Chain Nucleotide SequenceGAGGTCCAGCTCGTGGAAAGCGGAGGAGTGCTCGTGCAGCCTGGAGGCAGCCTCAGGCTGTCCTGTGCCGCCTCCGGCTTCACCTTCAGCAGCTACATCATGACCTGGGTGAGGCAGGCTCCCGGAAAAGGCCTGGAGTGGGTGTCCGGCATCTCCGGATCCGGAGGAGGCACATACTACGCCGACAGCATGAAGGGCCGGTTCACCATCAGCCGGGACAATAGCAAGAATACCCTCTACCTGCAAATGAACAGCCTGCGGGTGGAGGATACCGCCGTGTACTACTGCGCCAAAGATAGGCTGGGCCCCATTACCCTCGTGAGGGGAGGCTATTACTACGGCATGGATGTGTGGGGCCAGGGCACCACCGTGACAGTGTCCAGCGCCAGCACCAAGGGCCCTTCCGTGTTCCCCCTGGCCCCTTGCAGCAGGAGCACCTCCGAATCCACAGCTGCCCTGGGCTGTCTGGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCTCTGACATCCGGCGTCCACACCTTTCCTGCCGTCCTGCAGTCCTCCGGCCTCTACTCCCTGTCCTCCGTGGTGACCGTGCCTAGCTCCTCCCTCGGCACCAAGACCTACACCTGTAACGTGGACCACAAACCCTCCAACACCAAGGTGGACAAACGGGTCGAGAGCAAGTACGGCCCTCCCTGCCCTCCTTGTCCTGCCCCCGAGTTCGAAGGCGGACCCAGCGTGTTCCTGTTCCCTCCTAAGCCCAAGGACACCCTCATGATCAGCCGGACACCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTATGTGGATGGCGTGGAGGTGCACAACGCCAAGACAAAGCCCCGGGAAGAGCAGTTCAACTCCACCTACAGGGTGGTCAGCGTGCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCAGCAATAAGGGACTGCCCAGCAGCATCGAGAAGACCATCTCCAAGGCTAAAGGCCAGCCCCGGGAACCTCAGGTGTACACCCTGCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCTCCCGTCCTCGACAGCGACGGATCCTTCTTTCTGTACTCCAGGCTGACCGTGGATAAGTCCAGGTGGCAGGAAGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGAGCCTGTCCCTGGGAAAG 30 Heavy Chain Amino Acid SequenceEVQLVESGGVLVQPGGSLRLSCAASGFTFSSYIMTWVRQAPGKGLEWVSGISGSGGGTYYADSMKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCAKDRLGPITLVRGGYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 31 Light Chain Nucleotide SequenceGACATCCAGATGACCCAGTCCCCTTCCTCCCTGTCCGCCTCCGTGGGAGACAGGGTGACCATCACCTGCCGGGCCAGCCAGTCCATCAGCGACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGTTCCTGATCTACGCCGCTTCCTCCCTGCAGTCCGGAGTGCCCAGCAGGTTTTCCGGCTCCGGATCCGGCACCGACTTCACCCTGACCGTGTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAGGACATTTGGCCAGGGCACCCGGGTGGAGATCAAGAGGACCGTCGCTGCCCCCTCCGTGTTTATCTTCCCCCCCAGCGACGAGCAGCTGAAATCCGGCACCGCCTCCGTGGTCTGCCTGCTGAATAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAAACTCCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACTCCACATACTCCCTGTCCTCCACCCTGACACTGTCCAAGGCCGATTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCCTCCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGC 32 Light Chain Amino AcidSequence DIQMTQSPSSLSASVGDRVTITCRASQSISDYLNWYQQKPGKAPKFLIYAASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQSYSTPRTFGQGTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 3302D10 VH Nucleotide SequenceGAGGTGCAGTTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTTAGCAACTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGCGGAAGTGGTGGTGCCACAAGGTATGCAGACTCCGTGAAGGGCCGATTCACCATATCCAGAGACAATTCCAGGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTTTTTTACTGTACGAAAGATCGGCTCATTATGGCTACGGTTCGGGGACCCTATTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCCTCA 34 VH AminoAcid Sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWVSTISGSGGATRYADSVKGRFTISRDNSRNTVYLQMNSLRVEDTAVFYCTKDRLIMATVRGPYYYGMDVWGQGTTVTVSS 35 HCDR1 Nucleotide Sequence (IMGT)GGATTCACTTTTAGCAACTATGCC 36 HCDR1 Amino Acid Sequence (IMGT) GFTFSNYA 37HCDR2 Nucleotide Sequence (IMGT) ATTAGCGGAAGTGGTGGTGCCACA 38 HCDR2 AminoAcid Sequence (IMGT) ISGSGGAT 39 HCDR3 Nucleotide Sequence (IMGT)ACGAAAGATCGGCTCATTATGGCTACGGTTCGGGGACCCTATTACT ACGGTATGGACGTC 40 HCDR3Amino Acid Sequence (IMGT) TKDRLIMATVRGPYYYGMDV 41 HCDR1 NucleotideSequence (KABAT) AACTATGCCATGAAC 42 HCDR1 Amino Acid Sequence (KABAT)NYAMN 43 HCDR2 Nucleotide Sequence (KABAT)ACTATTAGCGGAAGTGGTGGTGCCACAAGGTATGCAGACTCCGTGA AGGGC 44 HCDR2 Amino AcidSequence (KABAT) TISGSGGATRYADSVKG 45 HCDR3 Nucleotide Sequence (KABAT)GATCGGCTCATTATGGCTACGGTTCGGGGACCCTATTACTACGGTAT GGACGTC 46 HCDR3 AminoAcid Sequence (KABAT) DRLIMATVRGPYYYGMDV 47 VL Nucleotide SequenceGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGAGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTCACAGTGTCTCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA 48 VL Amino Acid SequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSHSVSFTFG PGTKVDIK 49 LCDR1Nucleotide Sequence (IMGT) CAGAGCATTAGCAGCTAT 50 LCDR1 Amino AcidSequence (IMGT) QSISSY 51 LCDR2 Nucleotide Sequence (IMGT) GCTGCATCC 52LCDR2 Amino Acid Sequence (IMGT) AAS 53 LCDR3 Nucleotide Sequence (IMGT)CAACAGAGTCACAGTGTCTCATTCACT 54 LCDR3 Amino Acid Sequence (IMGT)QQSHSVSFT 55 LCDR1 Nucleotide Sequence (KABAT)CGGGCAAGTCAGAGCATTAGCAGCTATTTAAAT 56 LCDR1 Amino Acid Sequence (KABAT)RASQSISSYLN 57 LCDR2 Nucleotide Sequence (KABAT) GCTGCATCCAGTTTGCAAAGT58 LCDR2 Amino Acid Sequence (KABAT) AASSLQS 59 LCDR3 NucleotideSequence (KABAT) CAACAGAGTCACAGTGTCTCATTCACT 60 LCDR3 Amino AcidSequence (KABAT) QQSHSVSFT 61 Heavy Chain Nucleotide SequenceGAAGTGCAACTGGTGGAGTCCGGAGGAGGCCTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACTACGCCATGAACTGGGTGAGGCAGGCCCCTGGCAAGGGACTGGAGTGGGTCTCCACCATCAGCGGCTCCGGAGGCGCTACACGGTACGCCGATAGCGTGAAGGGCCGGTTTACCATTTCCCGGGACAACTCCCGGAACACCGTGTACCTCCAGATGAACAGCCTGAGGGTGGAGGATACCGCCGTGTTCTACTGCACCAAGGACAGGCTGATTATGGCCACCGTGAGGGGACCTTACTACTATGGCATGGATGTGTGGGGCCAGGGCACAACCGTCACCGTGTCCTCCGCCTCCACCAAGGGACCTAGCGTGTTCCCTCTCGCCCCCTGTTCCAGGTCCACAAGCGAGTCCACCGCTGCCCTCGGCTGTCTGGTGAAAGACTACTTTCCCGAGCCCGTGACCGTCTCCTGGAATAGCGGAGCCCTGACCTCCGGCGTGCACACATTTCCCGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTGGTGACCGTGCCCAGCTCCAGCCTCGGCACCAAAACCTACACCTGCAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGCGGGTGGAGAGCAAGTACGGCCCCCCTTGCCCTCCTTGTCCTGCCCCTGAGTTCGAGGGAGGACCCTCCGTGTTCCTGTTTCCCCCCAAACCCAAGGACACCCTGATGATCTCCCGGACACCCGAGGTGACCTGTGTGGTCGTGGACGTCAGCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCACAATGCCAAAACCAAGCCCAGGGAGGAGCAGTTCAATTCCACCTACAGGGTGGTGAGCGTGCTGACCGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGACTGCCCAGCTCCATCGAGAAGACCATCAGCAAGGCTAAGGGCCAGCCGAGGGAGCCCCAGGTGTATACCCTGCCTCCTAGCCAGGAAGAGATGACCAAGAACCAAGTGTCCCTGACCTGCCTGGTGAAGGGATTCTACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAATGGCCAGCCCGAGAACAACTACAAAACAACCCCTCCCGTGCTCGATAGCGACGGCAGCTTCTTTCTCTACAGCCGGCTGACAGTGGACAAGAGCAGGTGGCAGGAGGGCAACGTGTTCTCCTGTTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTCTCCCTGTCCCTGGGCAAG 62 Heavy Chain Amino Acid SequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWVSTISGSGGATRYADSVKGRFTISRDNSRNTVYLQMNSLRVEDTAVFYCTKDRLIMATVRGPYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 63 Light Chain Nucleotide SequenceGACATCCAGATGACCCAGTCCCCTTCCTCCCTGAGCGCTAGCGTGGGAGATAGGGTGACCATCACCTGCAGGGCCTCCCAAAGCATCTCCTCCTACCTGAACTGGTACCAGCAGAAACCCGGCAAGGCCCCCAACCTGCTGATCTACGCTGCCTCCTCCCTCCAGTCCGGCGTGCCTAGCAGGTTTAGCGGCTCCGGAAGCGAGACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAATCCCACAGCGTGTCCTTCACCTTCGGCCCCGGCACCAAGGTGGACATCAAGAGGACCGTGGCCGCCCCCTCCGTGTTCATCTTTCCCCCCTCCGATGAACAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCATCAGGGCCTGAGCTCCCCCGTGACCAAGTCCTTTAACAGGGGCGAGTGC 64 Light Chain Amino AcidSequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSHSVSFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 6509H04 VH Nucleotide SequenceCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTCGATTCACCCTCAGTGACTACTACATGACCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAATACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCTGAGTGGGAGCTACTGGGACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA 66 VH Amino AcidSequence QVQLVESGGGLVKPGGSLRLSCAASRFTLSDYYMTWIRQAPGKGLEWVSYISSSGNTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLSGSYWDYYYGMDVWGQGTTVTVSS 67 HCDR1 Nucleotide Sequence (IMGT)CGATTCACCCTCAGTGACTACTAC 68 HCDR1 Amino Acid Sequence (IMGT) RFTLSDYY 69HCDR2 Nucleotide Sequence (IMGT) ATTAGTAGTAGTGGTAATACCATA 70 HCDR2 AminoAcid Sequence (IMGT) ISSSGNTI 71 HCDR3 Nucleotide Sequence (IMGT)GCGAGAGATCTGAGTGGGAGCTACTGGGACTACTACTACGGTATGG ACGTC 72 HCDR3 Amino AcidSequence (IMGT) ARDLSGSYWDYYYGMDV 73 HCDR1 Nucleotide Sequence (KABAT)GACTACTACATGACC 74 HCDR1 Amino Acid Sequence (KABAT) DYYMT 75 HCDR2Nucleotide Sequence (KABAT)TACATTAGTAGTAGTGGTAATACCATATACTACGCAGACTCTGTGA AGGGC 76 HCDR2 Amino AcidSequence (KABAT) YISSSGNTIYYADSVKG 77 HCDR3 Nucleotide Sequence (KABAT)GATCTGAGTGGGAGCTACTGGGACTACTACTACGGTATGGACGTC 78 HCDR3 Amino AcidSequence (KABAT) DLSGSYWDYYYGMDV 79 VL Nucleotide SequenceGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTACATCTGTAGGAGACAGAGTCACCATCGCTTGCCGGGCAAGTCAGGGCATTAACAATGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 80 VL Amino Acid SequenceAIQLTQSPSSLSTSVGDRVTIACRASQGINNALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPRTFG QGTKVEIK 81 LCDR1Nucleotide Sequence (IMGT) CAGGGCATTAACAATGCT 82 LCDR1 Amino AcidSequence (IMGT) QGINNA 83 LCDR2 Nucleotide Sequence (IMGT) GATGCCTCC 84LCDR2 Amino Acid Sequence (IMGT) DAS 85 LCDR3 Nucleotide Sequence (IMGT)CAACAGTTTAATAGTTACCCTCGGACG 86 LCDR3 Amino Acid Sequence (IMGT)QQFNSYPRT 87 LCDR1 Nucleotide Sequence (KABAT)CGGGCAAGTCAGGGCATTAACAATGCTTTAGCC 88 LCDR1 Amino Acid Sequence (KABAT)RASQGINNALA 89 LCDR2 Nucleotide Sequence (KABAT) GATGCCTCCAGTTTGGAAAGT90 LCDR2 Amino Acid Sequence (KABAT) DASSLES 91 LCDR3 NucleotideSequence (KABAT) CAACAGTTTAATAGTTACCCTCGGACG 92 LCDR3 Amino AcidSequence (KABAT) QQFNSYPRT 93 19H01 VH Nucleotide SequenceGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGAAGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGATTTTCTATGGTTCGGGGAGTTCCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 94 VH Amino AcidSequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDFLWFGEFPFDYWGQGTLVTVSS 95 HCDR1 Nucleotide Sequence (IMGT)GGATTCACTTTCAGTAACGCCTGG 96 HCDR1 Amino Acid Sequence (IMGT) GFTFSNAW 97HCDR2 Nucleotide Sequence (IMGT) ATTAAAAGCAAAACTGAAGGTGGGACAACA 98 HCDR2Amino Acid Sequence (IMGT) IKSKTEGGTT 99 HCDR3 Nucleotide Sequence(IMGT) ACCACAGATTTTCTATGGTTCGGGGAGTTCCCTTTTGACTAC 100 HCDR3 Amino AcidSequence (IMGT) TTDFLWFGEFPFDY 101 HCDR1 Nucleotide Sequence (KABAT)AACGCCTGGATGAGC 102 HCDR1 Amino Acid Sequence (KABAT) NAWMS 103 HCDR2Nucleotide Sequence (KABAT)CGTATTAAAAGCAAAACTGAAGGTGGGACAACAGACTACGCTGCA CCCGTGAAAGGC 104 HCDR2Amino Acid Sequence (KABAT) RIKSKTEGGTTDYAAPVKG 105 HCDR3 NucleotideSequence (KABAT) GATTTTCTATGGTTCGGGGAGTTCCCTTTTGACTAC 106 HCDR3 AminoAcid Sequence (KABAT) DFLWFGEFPFDY 107 VL Nucleotide SequenceGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAATTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 108 VL Amino Acid SequenceDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKIPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPRTF GQGTKVEIK 109 LCDR1Nucleotide Sequence (IMGT) CAGGGCATTAGCAATTAT 110 LCDR1 Amino AcidSequence (IMGT) QGISNY 111 LCDR2 Nucleotide Sequence (IMGT) GCTGCATCC112 LCDR2 Amino Acid Sequence (IMGT) AAS 113 LCDR3 Nucleotide Sequence(IMGT) CAAAAGTATAACAGTGCCCCTCGGACG 114 LCDR3 Amino Acid Sequence (IMGT)QKYNSAPRT 115 LCDR1 Nucleotide Sequence (KABAT)CGGGCGAGTCAGGGCATTAGCAATTATTTAGCC 116 LCDR1 Amino Acid Sequence (KABAT)RASQGISNYLA 117 LCDR2 Nucleotide Sequence (KABAT) GCTGCATCCACTTTGCAATCA118 LCDR2 Amino Acid Sequence (KABAT) AASTLQS 119 LCDR3 NucleotideSequence (KABAT) CAAAAGTATAACAGTGCCCCTCGGACG 120 LCDR3 Amino AcidSequence (KABAT) QKYNSAPRT 121 Human IgG4 IGHG Heavy Chain ConstantRegion Nucleotidegcttccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgcccheavy chain *01 Sequencetgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggconstantcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagregion #1cagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaa 122 Heavy Chain Constant Region AminoASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Acid SequenceVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 123 Human IgG4 IGHG Heavy ChainConstant Region Nucleotidegcttccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgcccheavy chain *02 Sequencetgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggconstantcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagregion #2cagatgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccgtgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctctgggtaaa 124 Heavy Chain Constant Region AminoASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Acid SequenceVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 125 Human IgG4 IGHG Heavy ChainConstant Region Nucleotidegcttccaccaagggcccatccgtatccccctggcgccctgctccaggagcacctccgagagcacagccgcccheavy chain *03 Sequencetgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggconstantcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagregion #3cagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcaggggggaccatcagtatcagttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcaggaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctctgggtaaa 126 Heavy Chain Constant Region AminoASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Acid SequenceVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 127 IgG4 heavy Heavy Chain ConstantRegion Nucleotidegcctccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacggccgccchain constant Sequence - Synthetic Version Actgggctgcctggtcaaggactacttccccgaaccagtgacggtgtcgtggaactcaggcgccctgaccagcgregiongcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccaccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcgcctgaatttgaggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggatccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttacatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaa 128 IgG4 heavy Heavy Chain ConstantRegion Amino ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG chainconstant Acid Sequence - Encoded by SyntheticVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR region -IgG4- VersionA, B & C VESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS PEQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 129 IgG4 heavy Heavy Chain ConstantRegion NucleotideGcctccaccaagggacctagcgtgttccctctcgccccctgttccaggtccacaagcgagtccaccgctgccctchain constant Sequence - Synthetic Version Bcggctgtctggtgaaagactactttcccgagcccgtgaccgtctcctggaatagcggagccctgacctccggcgtregiongcacacatttcccgccgtgctgcagagcagcggactgtatagcctgagcagcgtggtgaccgtgcccagctccagcctcggcaccaaaacctacacctgcaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggagagcaagtacggccccccttgccctccttgtcctgcccctgagttcgagggaggaccctccgtgttcctgtttccccccaaacccaaggacaccctgatgatctcccggacacccgaggtgacctgtgtggtcgtggacgtcagccaggaggaccccgaggtgcagttcaactggtatgtggacggcgtggaggtgcacaatgccaaaaccaagcccagggaggagcagttcaattccacctacagggtggtgagcgtgctgaccgtcctgcatcaggattggctgaacggcaaggagtacaagtgcaaggtgtccaacaagggactgcccagctccatcgagaagaccatcagcaaggctaagggccagccgagggagccccaggtgtataccctgcctcctagccaggaagagatgaccaagaaccaagtgtccctgacctgcctggtgaagggattctacccctccgacatcgccgtggagtgggagagcaatggccagcccgagaacaactacaaaacaacccctcccgtgctcgatagcgacggcagcttctttctctacagccggctgacagtggacaagagcaggtggcaggagggcaacgtgttctcctgttccgtgatgcacgaggccctgcacaatcactacacccagaagagcctctccctgtccctgggcaag 130 IgG4 heavy Heavy Chain Constant RegionNucleotidegccagcaccaagggcccttccgtgttccccctggccccttgcagcaggagcacctccgaatccacagctgccctchain constant Sequence - Synthetic Version Cgggctgtctggtgaaggactactttcccgagcccgtgaccgtgagctggaacagcggcgctctgacatccggcregiongtccacacctttcctgccgtcctgcagtcctccggcctctactccctgtcctccgtggtgaccgtgcctagctcctccctcggcaccaagacctacacctgtaacgtggaccacaaaccctccaacaccaaggtggacaaacgggtcgagagcaagtacggccctccctgccctccttgtcctgcccccgagttcgaaggcggacccagcgtgttcctgttccctcctaagcccaaggacaccctcatgatcagccggacacccgaggtgacctgcgtggtggtggatgtgagccaggaggaccctgaggtccagttcaactggtatgtggatggcgtggaggtgcacaacgccaagacaaagccccgggaagagcagttcaactccacctacagggtggtcagcgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtacaagtgcaaggtcagcaataagggactgcccagcagcatcgagaagaccatctccaaggctaaaggccagccccgggaacctcaggtgtacaccctgcctcccagccaggaggagatgaccaagaaccaggtgagcctgacctgcctggtgaagggattctacccttccgacatcgccgtggagtgggagtccaacggccagcccgagaacaattataagaccacccctcccgtcctcgacagcgacggatccttctttctgtactccaggctgaccgtggataagtccaggtggcaggaaggcaacgtgttcagctgaccgtgatgcacgaggccctgcacaatcactacacccagaagtccctgagcctgtccctgggaaag 131 IgG4 heavy Heavy Chain Constant RegionNucleotidegcctccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacggccgccchain constant Sequence - Synthetic Version Dctgggctgcctggtcaaggactacttccccgaaccagtgacggtgtcgtggaactcaggcgccctgaccagcgregiongcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcgcctccagttgcggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggatccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaa 132 Heavy Chain Constant Region AminoASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Acid Sequence -encoded by Synthetic VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVersion D VESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 133 Human IgG1 Heavy ChainConstant Region Nucleotidegcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggcccheavy chain Sequencetgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggconstantcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagregioncagatgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcgcgggggcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccacccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 134 Heavy Chain Constant RegionAmino ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG Acid SequenceVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 135 Human Cκ IGKC Cκ LightChain Constant Regioncgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgconstant *01 Nucleotide Sequencetgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggregioncaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 136 Cκ Light Chain Constant Region AminoRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ Acid SequenceSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 137 HumanCκ IGKC Cκ Light Chain Constant Regioncgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgconstant *02 Nucleotide Sequencetgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaacregiontcccaggagagtgtcacagagcaggagagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgccggcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 138 Cκ Light Chain Constant Region AminoRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ Acid SequenceSGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQGLSSP VTKSFNRGEC 139 HumanCκ IGKC Cκ Light Chain Constant Regioncgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgconstant *03 Nucleotide Sequencetgcctgctgaataacttctatcccagagaggccaaagtacagcggaaggtggataacgccctccaatcgggtaaregionctcccaggagagtgtcacagagcaggagagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 140 Cκ Light Chain Constant Region AminoRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQRKVDNALQ Acid SequenceSGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 141 HumanCκ IGKC Cκ Light Chain Constant Regioncgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgconstant *04 Nucleotide Sequencetgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaacregiontcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaactctacgcctgcgaagtcacccatcagggcctgagacgcccgtcacaaagagcttcaacaggggagagtgt 142 Cκ Light Chain Constant Region AminoRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ Acid SequenceSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSP VTKSFNRGEC 143 HumanCκ IGKC Cκ Light Chain Constant Regioncgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgconstant *05 Nucleotide Sequencetgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaacregiontcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcaacaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagacgcccgtcacaaagagcttcaacaggggagagtgc 144 Cκ Light Chain Constant Region AminoRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ Acid SequenceSGNSQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 145 HumanCλ IGCλ1 Cλ Light Chain Constant Regioncccaaggccaaccccacggtcactctgttcccgccctcctctgaggagctccaagccaacaaggccacactagtconstant *01 Nucleotide Sequencegtgtctgatcagtgacttctacccgggagctgtgacagtggcttggaaggcagatggcagccccgtcaaggcggregiongagtggagacgaccaaaccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 146 Cλ Light Chain Constant Region AminoPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVK Acid SequenceAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECS 147 Human CλIGCλ1 Cλ Light Chain Constant Regionggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctgaggagctccaagccaacaaggccacconstant *02 Nucleotide Sequenceactagtgtgtctgatcagtgacttctacccgggagctgtgacagtggcctggaaggcagatggcagccccgtcaregionaggcgggagtggagaccaccaaaccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 148 Cλ Light Chain Constant Region AminoGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSP Acid SequenceVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTV EKTVAPTECS 149 Human CλIGCλ2 Cλ Light Chain Constant Regionggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctgaggagctccaagccaacaaggccacconstant *01 Nucleotide Sequence - Version Aactagtgtgtctgatcagtgacttctacccgggagctgtgacagtggcctggaaggcagatggcagccccgtcaregionaggcgggagtggagaccaccaaaccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 150 Cλ Light Chain Constant RegionggccagcctaaggccgctccttctgtgaccctgttccccccatcctccgaggaactgcaggctaacaaggccacNucleotide Sequence - Version Bcctcgtgtgcctgatcagcgacttctaccctggcgccgtgaccgtggcctggaaggctgatagctctcctgtgaaggccggcgtggaaaccaccaccccttccaagcagtccaacaacaaatacgccgcctcctcctacctgtccctgacccctgagcagtggaagtcccaccggtcctacagctgccaagtgacccacgagggctccaccgtggaaaagaccgtggctcctaccgagtgctcc 151 Cλ Light Chain Constant RegionggccagcctaaagctgcccccagcgtcaccctgtacctccctccagcgaggagctccaggccaacaaggccaNucleotide Sequence - Version Cccctcgtgtgcctgatctccgacttctatcccggcgctgtgaccgtggcttggaaagccgactccagccctgtcaaagccggcgtggagaccaccacaccctccaagcagtccaacaacaagtacgccgcctccagctatctctccctgacccctgagcagtggaagtcccaccggtcctactcctgtcaggtgacccacgagggctccaccgtggaaaagaccgtcgcccccaccgagtgctcc 152 Cλ Light Chain Constant Region AminoGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSP Acid Sequence - Encodedby Version A, B VKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTV & CEKTVAPTECS 153 Human Cλ IGCλ2 Cλ Light Chain Constant Regionggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacconstant *02 Nucleotide Sequenceactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaregionaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 154 Cλ Light Chain Constant Region AminoGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV Acid SequenceKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHIRSYSCQVTHEGSTVE KTVAPTECS 155 Human CλIGCλ Cλ Light Chain Constant Regioncccaaggctgccccctcggtcactctgttcccaccctcctctgaggagcttcaagccaacaaggccacactggtgconstant 3*01 Nucleotide Sequencetgtctcataagtgacttctacccgggagccgtgacagttgcctggaaggcagatagcagccccgtcaaggcgggregionggtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagttgcccctacggaatgttca 156 Cλ Light Chain Constant Region AminoPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA Acid SequenceGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKT VAPTECS 157 Human CλIGCλ Cλ Light Chain Constant Regionggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctgaggagcttcaagccaacaaggccacconstant 3*02 Nucleotide Sequenceactggtgtgtctcataagtgacttctacccggggccagtgacagttgcctggaaggcagatagcagccccgtcaaregionggcgggggtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacggaatgttca 158 Cλ Light Chain Constant Region AminoGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGPVTVAWKADSSPV Acid SequenceKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVE KTVAPTECS 159 Human CλIGCλ Cλ Light Chain Constant Regionggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctgaggagcttcaagccaacaaggccacconstant 3*03 Nucleotide Sequenceactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaregionaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 160 Cλ Light Chain Constant Region AminoGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV Acid SequenceKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVE KTVAPTECS 161 Human CλIGCλ Cλ Light Chain Constant Regionggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacconstant 3*04 Nucleotide Sequenceactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaregionaggcgggagtggagaccaccacaccaccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca 162 Cλ Light Chain Constant Region AminoGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV Acid SequenceKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS 163 Human CλIGCλ Cλ Light Chain Constant Regionggtcagcccaaggctgccccatcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacconstant 6*01 Nucleotide Sequenceactggtgtgcctgatcagtgacttctacccgggagctgtgaaagtggcctggaaggcagatggcagccccgtcaregionacacgggagtggagaccaccacaccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgttca 164 Cλ Light Chain Constant Region AminoGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSP Acid SequenceVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTV EKTVAPAECS 165 Human CλIGCλ Cλ Light Chain Constant Regionggtcagcccaaggctgccccatcggtcactctgttcccaccctcctctgaggagcttcaagccaacaaggccacconstant 7*02 Nucleotide Sequenceactggtgtgtctcgtaagtgacttctacccgggagccgtgacagtggcctggaaggcagatggcagccccgtcaregionaggtgggagtggagaccaccaaaccctccaaacaaagcaacaacaagtatgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaagctacagctgccgggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgctct 166 Cλ Light Chain Constant Region AminoGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSP Acid SequenceVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTV EKTVAPAECS 167Recombinant Nucleotide SequenceATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGG HumanCGTGCACAGCGATTACAAGGATGACGACGATAAGCGTATGAAACA OX40LGATCGAAGATAAAATTGAAGAGATCTTGAGCAAAATCTATCATATC (LeaderGAAAACGAAATTGCGCGTATCAAAAAGCTGATTGGCGAACGTGGC sequence,GGTGGCAGCGGTGGCGGTAGCGGCGGTGGCAGCCAGGTGTCCCACC IsoleucineGATACCCCAGGATCCAGTCCATCAAGGTCCAGTTCACCGAGTACAA Zipper andAAAGGAGAAGGGATTCATCCTGACCTCCCAAAAGGAGGACGAGAT FLAGCATGAAGGTGCAAAACAACTCCGTGATCATCAACTGCGACGGCTTC SequenceTACCTGATCTCCCTGAAGGGCTACTTCTCCCAGGAGGTGAACATCTC Included)CCTGCACTACCAGAAGGACGAGGAGCCCCTGTTCCAGCTGAAGAAGGTGAGGTCCGTGAATTCCCTGATGGTGGCCAGCCTGACCTACAAGGACAAGGTCTACCTGAACGTGACCACCGACAACACCAGCCTGGACGACTTCCATGTCAACGGCGGCGAGCTGATCCTGATCCATCAGAACCCC GGCGAGTTTTGCGTCCTG 168Amino Acid Sequence MGWSCIILFLVATATGVHSDYKDDDDKRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGGSGGGSGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQN PGEFCVL 169 RecombinantNucleotide Sequence ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGGRhesus CGTGCACAGCGATTACAAGGATGACGACGATAAGCGTATGAAACA OX40LGATCGAAGATAAAATTGAAGAGATCTTGAGCAAAATCTATCATATC (LeaderGAAAACGAAATTGCGCGTATCAAAAAGCTGATTGGCGAACGTGGC Sequence,GGTGGCAGCGGTGGCGGTAGCGGCGGTGGCAGCCAGGTGTCCCACC FLAG andAATACCCCAGGATCCAGTCCATCAAGGTCCAGTTCACCGAGTACAA IsoleucineAAAGGAGGAGGGATTCATCCTGACCTCCCAAAAGGAGGACGAGAT zipperCATGAAGGTGCAAAACAACTCCGTGATCATCAACTGCGACGGCTTC included)TACCTGATCTCCCTGAAGGGCTACTTCTCCCAGGAGGTGAACATCTCCCTGCACTACCAGAAGGACGAGGAGCCCCTGTTCCAGCTGAAGAAGGTGAGGTCCGTGAATTCCCTGATGGTGGCCAGCCTGACCTACAAGGACAAGGTCTACCTGAACGTGACCACCGACAACACCAGCCTGGACGACTTCCATGTCAACGGCGGCGAGCTGATCCTGATCCATCAGAACCCC GGCGAGTTTTGCGTCCTG 170Amino Acid Sequence MGWSCIILFLVATATGVHSDYKDDDDKRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGGSGGGSGGGSQVSHQYPRIQSIKVQFTEYKKEEGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQN PGEFCVL 171 RecombinantNucleotide Sequence ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGGHuman CGTGCACAGCCTGCATTGCGTGGGCGACACCTATCCCTCCAACGAC OX40RAGGTGCTGCCACGAGTGCAGGCCTGGAAACGGCATGGTGAGCAGG (LeaderTGCAGCCGGTCCCAGAATACCGTGTGTAGGCCCTGCGGCCCCGGCT Sequence andTTTACAACGACGTGGTGTCCTCCAAGCCCTGCAAGCCCTGCACATG Human FcGTGCAACCTGCGGTCCGGCAGCGAGAGGAAGCAGCTCTGCACAGCC SequenceACCCAGGACACCGTCTGTAGGTGTAGGGCTGGCACCCAGCCTCTGG included)ACTCCTACAAGCCCGGCGTGGATTGTGCTCCTTGCCCTCCCGGCCATTTCTCCCCTGGCGACAACCAGGCTTGCAAGCCCTGGACCAACTGTACCCTGGCCGGCAAGCATACACTGCAGCCTGCTTCCAACTCCTCCGACGCTATCTGCGAGGATAGGGACCCCCCTGCCACACAACCCCAGGAGACACAGGGCCCTCCTGCTAGGCCCATCACAGTCCAACCCACCGAAGCCTGGCCCAGGACATCCCAAGGCCCTTCCACCAGGCCTGTGGAAGTGCCTGGAGGAAGGGCTGTGGCCATTGAAGGTCGTATGGATGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCTGA 172 Amino Acid SequenceMGWSCIILFLVATATGVHSLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAIEGRMDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSP 173 CellExpressed Nucleotide SequenceATGGAGAGGGTGCAGCCCCTCGAGGAGAACGTGGGAAACGCCGCC OX40LAGGCCTAGGTTCGAGAGGAACAAGCTGCTGCTGGTGGCTTCCGTGA (CHO/MEF)TCCAAGGACTCGGCCTGCTGCTCTGCTTCACCTACATCTGCCTCCAC (LeaderTTCAGCGCCCTGCAGGTGTCCCACCGATACCCCAGGATCCAGTCCA sequenceTCAAGGTCCAGTTCACCGAGTACAAAAAGGAGAAGGGATTCATCCT included)GACCTCCCAAAAGGAGGACGAGATCATGAAGGTGCAAAACAACTCCGTGATCATCAACTGCGACGGCTTCTACCTGATCTCCCTGAAGGGCTACTTCTCCCAGGAGGTGAACATCTCCCTGCACTACCAGAAGGACGAGGAGCCCCTGTTCCAGCTGAAGAAGGTGAGGTCCGTGAATTCCCTGATGGTGGCCAGCCTGACCTACAAGGACAAGGTCTACCTGAACGTGACCACCGACAACACCAGCCTGGACGACTTCCATGTCAACGGCGGCGAGCTGATCCTGATCCATCAGAACCCCGGCGAGTTTTGCGTCCTGTAA 174 Amino Acid SequenceMERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFC 175 Cell Expressed Nucleotide SequenceATGTGCGTGGGGGCTCGGCGGCTGGGCCGCGGGCCGTGTGCGGCTC OX40 receptorTGCTCCTCCTGGGCCTGGGGCTGAGCACCGTGACGGGGCTCCACTG (HT1080)TGTCGGGGACACCTACCCCAGCAACGACCGGTGCTGCCACGAGTGCAGGCCAGGCAACGGGATGGTGAGCCGCTGCAGCCGCTCCCAGAACACGGTGTGCCGTCCGTGCGGGCCGGGCTTCTACAACGACGTGGTCAGCTCCAAGCCGTGCAAGCCCTGCACGTGGTGTAACCTCAGAAGTGGGAGTGAGCGGAAGCAGCTGTGCACGGCCACACAGGACACAGTCTGCCGCTGCCGGGCGGGCACCCAGCCCCTGGACAGCTACAAGCCTGGAGTTGACTGTGCCCCCTGCCCTCCAGGGCACTTCTCCCCAGGCGACAACCAGGCCTGCAAGCCCTGGACCAACTGCACCTTGGCTGGGAAGCACACCCTGCAGCCGGCCAGCAATAGCTCGGACGCAATCTGTGAGGACAGGGACCCCCCAGCCACGCAGCCCCAGGAGACCCAGGGCCCCCCGGCCAGGCCCATCACTGTCCAGCCCACTGAAGCCTGGCCCAGAACCTCACAGGGACCCTCCACCCGGCCCGTGGAGGTCCCCGGGGGCCGTGCGGTTGCCGCCATCCTGGGCCTGGGCCTGGTGCTGGGGCTGCTGGGCCCCCTGGCCATCCTGCTGGCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCA AGATCTGA 176 Amino AcidSequence MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 177 OX40L15B07 Amino Acid Sequenceof OX40L15B07 EVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTGEPREL (Seq ID No179 in WO2011/073180, TableVATITGGSSINYGDFVKGRFTISIDNAKNTVYLQMNNLKPEDTAVYYC A-1)NFNKYVTSRDTWGQGTQVTVSS 178 OX40L01B11 Amino Acid Sequence of OX40L01B11EVQLVESGGGLVQAGGSLRLSCVASGRSFSTYIMGWFRQAPGKEREFV (Seq ID No 180 inWO2011/073180, Table ATISRSGITIRSADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA-1) AGPYVEQTLGLYQTLGPWDYWGQGTQVTVSS 179 OX40L01E07 Amino Acid Sequenceof OX40L01E07 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSIYAKGWFRQAPGKEREF (Seq IDNo 181 in WO2011/073180, TableVAAISRSGRSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY A-1)CAAVGGATTVTASEWDYWGLGTQVTVSS 180 OX40L01E10 Amino Acid Sequence ofOX40L01E10 EVQLVESGGGLVQAGDSLRLSCAASGLTFSSFAMGWFRQAPGKEREF (Seq ID No182 in WO2011/073180, TableVAAISRSGYGTSEADSVRDRFIISRDNAKNTVTLHLSRLKPEDTAVYYC A-1)AAEHTLGRPSRSQINYLYWGQGTQVTVSS 181 OX40L18E09 Amino Acid Sequence ofOX40L18E09 EVQLVESGGGLVQAGGSLRLSCAASRNILSLNTMGWYRHAPGKPREL (Seq ID No183 in WO2011/073180, TableVARISSNSKTDYADSVKGRFTISRDNAKNTVLLQMNSLKPEDTGVYYC A-1)NLNVWRTSSDYWGQGTQVTVSS 182 OX40L19A07 Amino Acid Sequence of OX40L19A07EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIAWFRQAPGKEREG (Seq ID No 184 inWO2011/073180, Table VSRIKISNGRTTYAGSVKGRFTISSDNAKNTVYLQMNSLNAEDTAVYYA-1) CAADRSSLLFGSNWDRKARYDYWGQGTQVTVSS 183 OX40L19D08 Amino AcidSequence of OX40L19D08 EVQLVESGGGLVQAGASLRLSCAASGRRFISNYAMGWFRQAPGQERA(Seq ID No 185 in WO2011/073180, TableFVAAISRSGSITYYTDSVKGRFSISRDYAKSTVYLQMDNLKPEDTAVYY A-1)CAADGGAVRDLTTNLPDYWGRGTQVTVSS 184 OX40L075 Amino Acid Sequence ofOX40L075 (Seq EVQLVESGGGLVQPGGSLRLSCAASGRSFSTYIMGWFRQAPGKEREFV ID No 199in WO2011/073180, Table A-ATISRSGITTRSADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCA 2) AGPYVEQTLGLYQTLGPWDYWGQGTLVTVSS 185 OX40L024 Amino Acid Sequence of OX40L024 (SeqEVQLVESGGG LVQPGGSLRLSCAASG RTFSSIYAKGWFRQAPG KERE ID No 200 inWO2011/073180, Table A- FV 2) AAISRSG RSTSYADSVKG RFTISRD NAKNTVYLQMNSLRPEDTAVYYCAA VGGATTVTASEWDYWGLGTLVTVSS 186 OX40L025 Amino AcidSequence of OX40L025 (Seq EVQLVESGGG LVQPGGSLRLSCAASG RTFSSIYAKGWFRQAPGKERE ID No 201 in WO2011/073180, Table A- FV 2) AAISRSG RSTSYADSVKGRFTISRD NSKNTVYLQM NSLRPEDTAVYYCAA VGGATTVTASEWDYWGLGTLVTVSS 187OX40L026 Amino Acid Sequence of OX40L026 (Seq EVQLVESGGGLVQPGGSLRLSCAASG RTFSSIYAKGWFRQAPG KERE ID No 202 in WO2011/073180,Table A- FV 2) AAISRSG RSTSYADSVKG RFTISRD NAKNTVYLQM NSLRPEDTAVYYCAAVGGATTVTASEWDYWGQGTLVTVSS 188 OX40L027 Amino Acid Sequence of OX40L027(Seq EVQLVESGGG LVQPGGSLRLSCAASG RTFSSIYAKGWFRQAPG KERE ID No 203 inWO2011/073180, Table A- FV 2) AAISRSG RSTSYADSVKG RFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA VGGATTVTASEWDYWGQGTLVTVSS 189 OX40L028 Amino AcidSequence of OX40L028 (Seq DVQLVESGGG LVQPGGSLRLSCAASG RTFSSIYAKGWFRQAPGID No 204 in WO2011/073180, Table A- KEREFV 2) AAISRSG RSTSYADSVKGRFTISRD NAKNTVYLQM NSLRPEDTAVYYCAA VGGATTVTASEWDYWGLGTLVTVSS 190OX40L039 Amino Acid Sequence of OX40L039 (Seq DVQLVESGGGLVQPGGSLRLSCAASG RTFSSIYAKGWFRQAPG KERE ID No 205 in WO2011/073180,Table A- FV 2) AAISRSG RSTSYADSVKG RFTISRD NSKNTVYLQM NSLRPEDTAVYYCAAVGGATTVTASEWDYWGQGTLVTVSS 191 OX40L030 Amino Acid Sequence of OX40L030(Seq DVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No 206 inWO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISID NAKNTVYLQM N N 2)LKPEDTAVYYCN FN KYVTSRDTWGQGTQVTVSS 192 OX40L040 Amino Acid Sequence ofOX40L040 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No 207in WO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 193 OX40L041 Amino Acid Sequenceof OX40L041 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHATGEPRELV ID No208 in WO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 194 OX40L042 Amino Acid Sequenceof OX40L042 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELV ID No209 in WO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 195 OX40L043 Amino Acid Sequenceof OX40L043 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGKPRELV ID No210 in WO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 196 OX40L044 Amino Acid Sequenceof OX40L044 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHAPGEPRELV ID No211 in WO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 197 OX40L045 Amino Acid Sequenceof OX40L045 (Seq DVQLVESGGG LVQPGGSLRLSCAASRSIG RLD RMGWYRHATG KPRE IDNo 212 in WO2011/073180, Table A- LV 2) ATITGGSSINYG D FVKGRFTISRDNSKNTVYLQM NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 198 OX40L046Amino Acid Sequence of OX40L046 (SeqDVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGKPRELV ID No 213 inWO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 199 OX40L047 Amino Acid Sequenceof OX40L047 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHAPGKPRELV ID No214 in WO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 200 OX40L048 Amino Acid Sequenceof OX40L048 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No215 in WO2011/073180, Table A- ATITGGSSINYADFVKG RFTISRD NSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 201 OX40L049 Amino Acid Sequenceof OX40L049 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No216 in WO2011/073180, Table A- ATITGGSSINYG DSVKG RFTISRDNSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 202 OX40L050 Amino Acid Sequenceof OX40L050 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No217 in WO2011/073180, Table A- ATITGGSSINYADSVKG RFTISRD NSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 203 OX40L053 Amino Acid Sequenceof OX40L053 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No218 in WO2011/073180, Table A- ATITGGSSINYGDFVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCN 2) FNK YVTS RDTWGQGTLVTVSS 204 OX40L054 Amino AcidSequence of OX40L054 (SeqDVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No 219 inWO2011/073180, Table A- ATITGGSSINYG D FVKG RFTISRDNAKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 205 OX40L055 Amino Acid Sequenceof OX40L055 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELV ID No220 in WO2011/073180, Table A- ATITGGSSINYGDFVKGRFTISRDNSKNTVYLQM NN 2)LRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 206 OX40L056 Amino Acid Sequence ofOX40L056 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGKPRELV ID No 221in WO2011/073180, Table A- ATITGGSSINYADSVKG RFTISRD NSKNTVYLQM 2)NSLRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 207 OX40L069 Amino Acid Sequenceof OX40L069 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGKPRELV ID No222 in WO2011/073180, Table A- ATITGGSSINYADSVKG RFTISI DNSKNTVYLQMNSLRPE 2) DTAVYYCN FN K YVTS RDTWGQGTLVTVSS 208 OX40L070 Amino AcidSequence of OX40L070 (SeqDVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGKPRELV ID No 223 inWO2011/073180, Table A- ATITGGSSINYADSVKG RFTISRD NSKNTVYLQM N N 2)LRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 209 OX40L071 Amino Acid Sequence ofOX40L071 (Seq DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGKPRELV ID No 224in WO2011/073180, Table A- ATITGGSSINYADSVKG RFTISI DNSKNTVYLQM N N 2)LRPEDTAVYYCN FN KYVTSRDTWGQGTLVTVSS 210 OX40L082 Amino Acid Sequence ofOX40L082 (Seq EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVA ID No225 in WO2011/073180, Table A- TITGGSSINYGDSVKGRFTISIDNSKNTVYLQM 2)NSLRPEDTAVYYCNFNKY VTS RDTWGQGTLVTVSS 211 OX40L083 Amino Acid Sequenceof OX40L083 (Seq EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGKPRELV ID No226 in WO2011/073180, Table A-ATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCN 2) FNK YVTSRDTWGQGTLVTVSS 212 OX40L Amino acid sequence of OX40LEVQLLESGGGLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEW benchmark benchmarkantibody heavy chain (Seq ID VSlISGSGG FTYYADSVKG RFTISRDNSRTTLYLQMantibody No: 177 in WO2011/073180, Table A-5) NSLRAEDTAVYYCA heavy chainKDRLVAPGTFDYWGQGALVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF P PKPKDTLM IS RTPEVTCVVVDVSH E DP EVKFNWYVDGVEVH NAKTKP REEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSN KALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQG NVFSCSVM H EALH N HYTQ KSLSLSPGK 213OX40L Amino acid sequence of OX40LDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIY benchmark benchmarkantibody light chain (Seq ID AASSLQSGVPSRFSGSGSGTDFTLTISSLQPED antibodylight No: 178 in WO2011/073180, Table A-5) FATYYCQQYNSYPYTFG chainQGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLN N FYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTH QGLSSPVTKSFNRGEC 214 κ lightchain Amino acid sequence of kappa light chainDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIY variable regionvariable region of LC.001 (Seq ID No: 1 inAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFG of LC.001WO2006/029879) QGTKLEIK 215 γ heavy chain Amino acid sequence of γ heavychain EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEW variable regionvariable region of LC.001 (Seq ID No: 2 inVSIISGSGGFTYYADSVKGRFTISRDNSRTTLYLQMNSLRAEDTAVYYC of LC.001WO2006/029879) AKDRLVAPGTFDYWGQGALVTVSS 216 κ light chain Amino acidsequence of kappa light chainEIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLI variable regionvariable region of LC.005 (Seq ID No: 3 inYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFTFG of LC.005WO2006/029879) PGTKVDIK 217 γ heavy chain Amino acid sequence of γ heavychain QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFGMHWVRQAPGKGLE variable regionvariable region of LC.005 (Seq ID No: 4 inWVAAIWYDGHDKYYSYYVKGRFTISRDNSKNTLFLQMNSLRAEDTAV of LC.005 WO2006/029879)YYCARDSSSWYRYFDYWGQGTLVTVSS 218 κ light chain Amino acid sequence ofkappa light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYvariable region variable region of LC.010 (Seq ID No: 5 inGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFTFGP of LC.010WO2006/029879) GTKVDIK 219 γ heavy chain Amino acid sequence of γ heavychain QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFGMHWVRQAPGKGLE variable regionvariable region of LC.010 (Seq ID No: 6 inWVAAIWYDGHDKYYAYYVKGRFTISRDNSKNTLFLQMNSLRAEDTA of LC.010 WO2006/029879)VYYCARDSSSWYRYFDYWGQGTLVTVSS 220 κ light chain Amino acid sequence ofkappa light chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFGMHWVRQAPGKGLEvariable region variable region of LC.029 (Seq ID No: 7 inWVAAIWYDGHDKYYSYYVKGRFTISRDNSKNTLFLQMNSLRAEDTAV of LC.029 WO2006/029879)YYCARDSSSWYRYFDYWGQGTLVTVSS 221 γ heavy chain Amino acid sequence ofγ heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEW variableregion variable region of LC.029 (Seq ID No: 8 inVAVIWYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY of LC.029 WO2006/029879)YCARKNWSFDFWGQGTLVTVSS 222 κ light chain Amino acid sequence of kappalight chain EIVLTQSPATLSLSPGERATLSCRASQGVSRYLAWYQQKPGQAPRLLIY variableregion variable region of LC.019 (Seq ID No: 9 inDASNRATGIPARVSGSGPGTDFTLTISSLEPEDFAVDYCQQRSNWQYTF of LC.019WO2006/029879) GQGTKLEI 223 γ heavy chain Amino acid sequence of γ heavychain QKQLVEFGGGVVQPGRSLRLSCAASGFTFSNYGMIHWVRQAPGKGLE variable regionvariable region of LC.019 (Seq ID No: 10WVAVIWNDGSNKYYVDSVKGRFIISRDNSKNTLYLQMNSLRAEDTAV of LC.019 inWO2006/029879) YYCARDRMGIYYYGMDVWGQGTTVTVSS 224 κ light chain Amino acidsequence of kappa light chainEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY variable regionvariable region of LC.033 (Seq ID No: 11DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWTFGQ of LC.033 inWO2006/029879) GTKVEI 225 γ heavy chain Amino acid sequence of γ heavychain EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEW variable regionvariable region of LC.033 (Seq ID No: 12VSIISGSGGFTYYADSVKGRFTISRDNSRTTLYLQMNSLRAEDTAVYYC of LC.033 inWO2006/029879) AKDRLVAPGTFDYWGQGALVTVSS 226 mutant κ light Amino acidsequence of mutant kappaEIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLI chain variable lightchain variable region of LC.033 (SeqYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFTFG region of ID No: 16in WO2006/029879) PGTKVDIK LC.033 227 γ heavy chain Amino acid sequenceof γ heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWvariable region variable region of LC.059 (Seq ID No: 17VSIISGSGGFTYYADSVKGRFTISRDNSKNTLYLQMNRLRAEDTAIYFC of LC.059 inWO2006/029879) AKDDIPAAGTFDPWGQGTLVTVSS 228 κ light chain Amino acidsequence of kappa light chainAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIY variable regionvariable region of LC.060 (Seq ID No: 18DVSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYWTFG of LC.060 inWO2006/029879) QGTKVEIK 229 γ heavy chain Amino acid sequence of γ heavychain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW variable regionvariable region of LC.060 (Seq ID No: 19VSLISGSGGLTKYADSVKGRFTISRDNSKRTLYLQMNSLRAEDTAVYY of LC.060 inWO2006/029879) CAKDILVTGALDYWGQGTLVTVSS 230 γ heavy chain Amino acidsequence of γ heavy chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEW variable region variableregion of LC.063 (Seq ID No: 20VSIISGSGGFTYYADSVKGRFTISRDNSKKTLYLQMSRLRAEDTAIYFC of LC.063 inWO2006/029879) AKDDIPAAGTFDPWGQGTLVTVSS 231 8E12 light Amino acidsequence of 8E12 light chainDILMTQTPLSLPVSLGDQASISCRSSQSIVHGNGNTYLEWHLQKPGQSP chain variablevariable region (Seq ID No: 13 inKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCFQGSH region U.S. Pat. No.7,812,133) VPYTFGGGTKVEIKR 232 8E12 heavy Amino acid sequence of 8E12heavy chain DIVMTQTPLSLPVSLGDQASMYCRSSQSPVHSNGNTYLHWYLQKPGQS chainvariable variable region (Seq ID No: 14 inPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTH region U.S. Pat. No.7,812,133) IPWTFGGGTKVEIKR 233 13G5 light Amino acid sequence of 13G5light chain QVQLQQPGAELVRPGASVkLSCKASGYTFTSYWLNWVKQRPGQGLE chainvariable variable region (Seq ID No: 15 inWIVMIDPSDSETHYNQVFKDKATLTVDKSSSTAYMQLSSLTSEDSAVY region U.S. Pat. No.7,812,133) YCIRGRGNEYGGSHAMEYWGQGTLLTVSS 234 13G5 heavy Amino acidsequence of 13G5 heavy chainQVQLQQPGAELVKPGTSVKLSCKASGYSFTSYWMHGVRQRPGQGLE chain variable variableregion (Seq ID No: 16 inWIGEIDPSNGRTNYNEKFKSKATLTVDKSSSTAYIQLSSLTSEDSAVYY region U.S. Pat. No.7,812,133) CTRERSPRYFDVWGAGTTLTVSS IMGT indicates that CDR is determinedusing IMGT nomenclature; KABAT indicates that CDR is determined usingKabat nomenclature. The numbering in the sequence correlation tabletakes precedence over any inconsistent numbering elsewhere in this text.

1-27. (canceled)
 28. A method of treating or preventing transplantrejection in a human subject in need thereof, comprising: administeringto the subject a therapeutically or prophylactically effective amountof: a) at least one anti-OX40L antibody or fragment thereof thatantagonizes specific binding of OX40 to OX40L; and b) at least oneCTLA4-Fc.
 29. The method of claim 28, wherein the CTLA4-Fc is abatacept.30. The method of claim 28, wherein the anti-OX40L antibody or fragmentthereof is administered at least once before a transplant.
 31. Themethod of claim 28, wherein the anti-OX40L antibody or fragment thereofis administered at least once after a transplant.
 32. The method ofclaim 28, wherein the CTLA4-Fc is administered at least once after atransplant.
 33. The method of claim 28, wherein the CTLA4-Fc isadministered at least once before a transplant.
 34. The method of claim28, wherein the anti-OX40L antibody or fragment thereof is administeredat least once before a transplant and at least once after thetransplant.
 35. The method of claim 28, wherein the CTLA4-Fc isadministered at least once before a transplant and at least once afterthe transplant.
 36. The method of claim 28, wherein the anti-OX40Lantibody or fragment thereof is administered at least once before atransplant and the CTLA4-Fc is administered at least once after thetransplant.
 37. The method of claim 28, wherein the CTLA4-Fc isadministered at least once before a transplant and the anti-OX40Lantibody or fragment thereof is administered at least once after thetransplant.
 38. The method of claim 28, wherein the anti-OX40L antibodyor fragment thereof and the CTLA4-Fc are administered at least oncebefore transplant.
 39. The method of claim 28, wherein the anti-OX40Lantibody or fragment thereof and the CTLA4-Fc are administered at leastonce after transplant.
 40. The method of claim 28, wherein theadministration of the anti-OX40L antibody or fragment thereof and theadministration of the CTLA4-Fc are continued at least until peripheralblood chimerism has been achieved in the human subject.
 41. The methodof claim 28, wherein the anti-OX40L antibody or fragment thereof isadministered at least once before a transplant followed by once a week,bi-weekly or once a month administration of the same after thetransplant; and the CTLA4-Fc is administered at least once after thetransplant.
 42. The method of claim 28, wherein the anti-OX40L antibodyor fragment thereof is administered at least once before a transplantfollowed by once a week, bi-weekly or once a month administration of thesame after the transplant; and the CTLA4-Fc is administered daily afterthe transplant.
 43. The method of claim 28, wherein the anti-OX40Lantibody or fragment thereof is a humanized or human antibody orantibody fragment.
 44. The method of claim 28, wherein the anti-OX40Lantibody or fragment thereof is selected from the group consisting of:multi-specific antibodies, bi-specific antibodies, single chain Fvantibodies (scFv), camelized antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), and epitope-binding fragmentsthereof.
 45. The method of claim 28, wherein the anti-OX40L antibody orfragment thereof comprises a HCDR3 of from 16 to 27 amino acids andderived from the recombination of a human VH gene segment, a human Dgene segment and a human JH gene segment, wherein the human JH genesegment is IGHJ6 or IGHJ6*02.
 46. The method of claim 28, wherein theanti-OX40L antibody or fragment thereof comprises a CDR selected from:a. the HCDR3 of antibody comprising variable region Seq ID No:40 or SeqID No:46; b. the HCDR3 of antibody comprising variable region Seq IDNo:8 or SEQ ID No:14; c. the HCDR3 of antibody comprising variableregion Seq ID No:72 or Seq ID No:78; d. the HCDR3 of antibody comprisingvariable region Seq ID No:100 or Seq ID No:106; e. an HCDR3 of any ofthe antibodies having the variable region amino acid sequence of Seq IDNos: 215, 217, 219, 221, 223, 225, 227, 229 or 230; and f. an HCDR3 ofany of the antibodies having the variable region amino acid sequence ofSeq ID Nos: 232 or
 234. 47. The method of claim 28, wherein theanti-OX40L antibody or fragment thereof comprises: a. the CDRs of Seq IDNo:40 or Seq ID No:46 for CDRH3, SEQ ID No:38 or SEQ ID No:44 for CDRH2,SEQ ID No:36 or SEQ ID No:42 for CDRHI, SEQ ID No:50 or SEQ ID No:56 forCDRL1, SEQ ID No:52 or SEQ ID No:58 for CDRL2 and SEQ ID No:54 or SEQ IDNo:60 for CDRL3; b. the CDRs of Seq ID No:8 or SEQ ID No:14 for CDRH3,SEQ ID No:6 or SEQ ID No:12 for CDRH2, SEQ ID No:4 or SEQ ID No:10 forCDRH1, SEQ ID No:18 or SEQ ID No:24 for CDRL1, SEQ ID No:20 or SEQ IDNo:26 for CDRL2 and SEQ ID No:22 or SEQ ID No:28 for CDRL3; c. the CDRsof Seq ID No:72 or Seq ID No:78 for CDRH3, SEQ ID No:70 or SEQ ID No:76for CDRH2, SEQ ID No:68 or SEQ ID No:74 for CDRH1, SEQ ID No:82 or SEQID No:88 for CDRL1, SEQ ID No:84 or SEQ ID No:90 for CDRL2 and SEQ IDNo:86 or SEQ ID No:92 for CDRL3; d. the CDRs of Seq ID No:100 or Seq IDNo:106 for CDRH3, SEQ ID No:98 or SEQ ID No:104 for CDRH2, SEQ ID No:96or SEQ ID No: 102 for CDRH1, SEQ ID No:110 or SEQ ID No:116 for CDRL1,SEQ ID No:112 or SEQ ID No:118 for CDRL2 and SEQ ID No:114 or SEQ IDNo:120 for CDRL3; e. the heavy chain CDRs and the light chain CDRs ofvariable region amino acid sequences SEQ ID NO: 215 and SEQ ID NO: 214;SEQ ID NO: 217 and SEQ ID NO: 216; SEQ ID NO: 219 and SEQ ID NO: 218;SEQ ID NO: 221 and SEQ ID NO: 220; SEQ ID NO: 223 and SEQ ID NO: 222;SEQ ID NO: 225 and SEQ ID NO: 224; SEQ ID NO: 225 and SEQ ID NO: 226;SEQ ID NO: 227 and SEQ ID NO: 214; SEQ ID NO: 229 and SEQ ID NO: 228; orSEQ ID NO: 230 and SEQ ID NO: 214; f. the heavy chain CDRs and the lightchain CDRs of variable region amino acid sequences SEQ ID NO: 232 andSEQ ID NO: 231; or SEQ ID NO: 234 and SEQ ID NO: 233; g. the VH and/orVL domains of Seq ID No:34 for VH and/or Seq ID No:48 for VL; h. the VHand/or VL domains of Seq ID No:2 for VH and/or Seq ID No:16 for VL; i.the VH and/or VL domains of Seq ID No:66 for VH and/or Seq ID No:80 forVL; j. the VH and/or VL domains of Seq ID No:94 for VH and/or Seq IDNo:108 for VL; k. the VH domain and the VL domain of amino acidsequences SEQ ID NO: 215 and SEQ ID NO: 214; SEQ ID NO: 217 and SEQ IDNO: 216; SEQ ID NO: 219 and SEQ ID NO: 218; SEQ ID NO: 221 and SEQ IDNO: 220; SEQ ID NO: 223 and SEQ ID NO: 222; SEQ ID NO: 225 and SEQ IDNO: 224; SEQ ID NO: 225 and SEQ ID NO: 226; SEQ ID NO: 227 and SEQ IDNO: 214; SEQ ID NO: 229 and SEQ ID NO: 228; or SEQ ID NO: 230 and SEQ IDNO: 214: or l. the VH domain and the VL domain of amino acid sequencesSEQ ID NO: 232 and SEQ ID NO: 231; or SEQ ID NO: 234 and SEQ ID NO: 233.48. The method of claim 28, wherein the anti-OX40L antibody or fragmentthereof comprises the VH sequence of SEQ ID No:215 and the VL sequenceof SEQ ID No:214.
 49. The method of claim 28, wherein the anti-OX40Lantibody or fragment thereof is oxelumab.
 50. The method of claim 28,wherein the anti-OX40L antibody or fragment thereof is administered atleast once before a transplant and at least once after the transplantand the CTLA4-Fc is administered at least once before a transplant andat least once after the transplant.